Patent Publication Number: US-11030531-B2

Title: DNA recombinase circuits for logical control of gene expression

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a 371 National Phase Entry of International Application PCT/US15/34721 filed on Jun. 8, 2015 which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 62/008,952 filed Jun. 6, 2014, and 62/144,678 filed Apr. 8, 2015, the contents of each of which are incorporated herein by reference in their entirety. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with Government Support under Contract Nos. CA186574 and GM008764 awarded by the National Institutes of Health and Contract No. DGE1247312 awarded by the National Science Foundation. The Government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The sequence listing of the present application has been submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “701586-081803-PCT_SL” creation date of Dec. 5, 2016 and a size of 227,944 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety. 
     TECHNICAL FIELD 
     The present invention relates generally to controlled gene expression, engineered gene circuits, and adoptive T cell therapies. 
     BACKGROUND OF THE INVENTION 
     Synthetic genetic circuits hold vast potential at revolutionizing therapeutics, animal models 2 , and biotechnological processes 3-6 . Despite rapid advances in recent years, engineering complex genetic circuits remains a challenge due to difficult circuit performance predictability stemming from unintended interactions between cascading biological components 7 , such as transcription factors 8-12 , chaperonins 13 , or miRNAs 14-17 . 
     A major goal in synthetic biology is to predictably design and construct genetic circuits to control cellular functions. Numerous success, such as logic gates, toggle switches, feedback loops and oscillators, has been demonstrated. Due to the complexity of mammalian systems, sophisticated genetic circuits that can integrate multiple input signals and deliver multiple outputs are highly desirable. For instance, the specificity of tissue-restricted gene expression can be greatly enhanced with the logical integration of several tissue-specific promoters. However, synthetic biology remains mostly a microbial centric discipline and high performance genetic circuits are lacking in mammalian cells. Furthermore, complex multi-input/multi-output genetic circuits are challenging to engineer, regardless of the host. 
     A common strategy for restricting gene expression in a specific cell type is to identify a single cell type specific promoter and use it to control recombinase expression. Although some cell type specific promoters have been identified, they do not cover the diversity represented by all the different cell types in complex organs, such as the mammalian brain. It is, however, much more likely to identify a set of promoters that collectively define a specific subset of cells rather than relying on one promoter. Extensive gene expression profiling in different brain regions and cells had allowed researchers to identify promoters that can define different type of brain cells. 
     A central goal of synthetic biology is to create cellular networks that integrate input signals for decision making and actuation. In recent years, artificial logic gates and memory devices have been independently constructed. In previous implementations of cellular logic, complex gates required the layering of multiple genetic circuits, thus necessitating significant efforts for circuit construction and tuning. These complex logic gates can achieve only combinatorial logic. 
     SUMMARY OF THE INVENTION 
     Synthetic genetic circuits that can integrate multiple input signals and deliver multiple outputs are increasingly important in cell-based therapy and animal model development. However, synthetic biology remains mostly a microbial centric discipline and complex genetic circuits remain challenging to engineer, regardless of the host. 
     Provided herein are strategies and genetic tools to incorporate input to regulate gene expression—genetic logic gates that can integrate different recombinase inputs in a single layered platform (e.g., a nucleic acid construct). The invention provides, inter alia, synthetic recombinase-based systems for integrating combinatorial logic and memory in living cells. Integrated logic and memory are crucial for performing complex and persistent state-dependent computation such as sequential logic. Using a defined set of programming rules, the logic and memory systems of the invention enable efficient, one-step assembly of any Boolean logic function with stable, DNA-based memory of events. These systems utilize chemical inducer as inputs to drive the expression of orthogonal recombinases from promoters. These recombinases target genetic elements for DNA inversion (e.g., reversible), or excision (e.g., irreversible), resulting in conditional nucleic acid expression. Such logic and memory systems are useful for a variety of applications, including programming cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications. 
     Some of the most powerful tools in genome engineering are site-specific DNA recombinases (SSR), which can be used to activate or inhibit expression of genes. One of the main functions of recombinases in genome engineering is to regulate gene expression, both for exogenous and endogenous genes. 
     The methodology of constructing complex nucleic acid logic cassettes described herein takes advantage of the simple operation principles of site-specific recombinases. As shown in  FIG. 11A , when a nucleic acid sequence (e.g., a target gene) is flanked by a pair of recombinase recognition sequences (RRS) in the same orientation, the nucleic acid sequence can be excised upon the recognition of the RRS by the proper recombinase; when a nucleic acid sequence is flanked by a pair of recombinase recognition sequences (RRS) in an inverse orientation, the nucleic acid sequence can be inverted upon the recognition of the RRS by the proper recombinase. The inversion and/or excision reactions will place the nucleic acid sequence (e.g., a target gene) in a new location and/or orientation to make it operatively linked to a promoter, thereby driving expression of the nucleic acid sequence. Based on these operation principles, depending on the combination of excision and inversion of 1, 2, or more nucleic acid sequence(s), one can select from the growing number of recombinases and their corresponding RRS to construct nucleic acid logic cassettes for a specific logic function. 
     Depending on the location of the recombinase recognition sequences, the excision reaction by a recombinase can lead to activation or inhibition of gene expression. For example, to activate gene expression, one can put a transcription termination sequence, flanked on both sides by recombination sites, upstream of a target gene of interest. In the presence of the recombinase, the transcription termination sequence is removed, thus allowing gene expression to occur. In contrast, to inhibit gene expression, the recombination sites can be engineered to flank the gene of interest. The gene expresses without the recombinase. Once the recombinase is induced, the gene will be excised and thus the gene expression is completely and irreversibly inhibited. The determinant factor that governs the on/off state of the gene resides in the presence of the recombinase. The expression of the recombinase can be regulated by a signal inducible promoter, thus restricting the expression of the recombinase in a certain subset of cells that are undergoing that particular signal. For instance, by controlling the expression of Cre recombinase with neuron specific promoters, one can turn genes on or off in neurons only while leaving the same gene unperturbed in other tissues. These enzymes have been engineered to be very active in a wide range of organisms, including bacteria, mammals, insects, plants and fish. Due to their ability to modify DNA in almost any tissues and conditions, recombinases have proven to be invaluable in regulating gene expression for many studies. 
     However, most existing recombinase-based expression platform utilizes one, or at most two recombinase. Therefore, these systems have limited capacity to perform complex and high level signal integration and computation. 
     Recently, orthogonal site-specific DNA recombinases (SSR) have been identified. It is therefore possible to use such orthogonal recombinases to perform complex logic computation. By coupling the expression of the recombinase with cell-type or condition specific promoters, the inventors have demonstrated robust spatiotemporal control of gene expression in living animals (e.g., mammals) in vivo. 
     Using these newly discovered recombinases, the inventors demonstrate herein sophisticated genetic logic gates, such as a Full Adder, Multi-Input (e.g., 3, 4, 6 and 8 inputs) AND gate, and Decoder. Furthermore, the inventors have developed a highly robust and modular genetic circuit platform in living human cells wherein the majority of computation is achieved by a single transcriptional unit, which can be rapidly and intuitively designed and constructed. Using the platform, the inventors have developed a large number of complex multi-input, multi-output circuits, including, but not limited to, (i) a 6-input AND gate, (ii) 2-input decoder, (iii) half adder, (iv) half subtractor, (v) Feynman gate, (vi) full adder, (vii) full subtractor, as well as (viii) two field-programmable genetic devices: a half adder-subtractor and Boolean Logic Look-Up Table. Accordingly, the inventors have created all sixteen two-input Boolean logic gates to regulate expression of a gene of interest. Gene circuits that can perform more complex logic functions can be created from a combination of these logic gates. Additionally, the inventors have created biological computers that utilize these recombinases to permit very fined-tuned and logic control of exogenous and endogenous genes. 
     To demonstrate the robustness of this platform, the inventors demonstrate the successful engineering of a variety of functionally distinct logic gates, and developed a quantitative metric to objectively assess the performance of the circuits. Given the importance of DNA recombinases in animal genetics 2,18,19  and the simplicity of the single transcriptional unit design disclosed herein, this platform can greatly improve tissue-specific gene expression and thus, the complexity of animal models available for studying diseases and drug development. 
     Furthermore, the inventors demonstrate that in contrast to prior genetic circuits, all of the circuits described herein are created in single layer and single transcription unit (e.g., in a single nucleic acid cassette or construct). Importantly, multiple plasmids are not necessary, and no linkages are needed between different transcriptional units in order to achieve the desired function. Thus the inventors have developed a modular genetic circuit platform that overcomes one of the most difficult challenges in synthetic circuit design—connecting various transcription units, and greatly accelerated the circuit development. 
     In one aspect, the invention relates to a nucleic acid logic cassette comprising: (i) a nucleic acid sequence encoding a promoter; (ii) a first recombination unit (U1) comprising a first pair of recombinase recognition sequences (RRS1) for a first site-specific recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter, whereby when the R1 recognizes the RRS1, the first nucleic acid sequence is excised when the RRS1 are in the same orientation or is inverted when the RRS1 are in the inverse orientation; (iii) a second recombination unit (U2) comprising a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1, whereby when the R2 recognizes the RRS2, the second nucleic acid sequence is excised when the RRS2 are in the same orientation or is inverted when the RRS2 are in the inverse orientation; (iv) a third recombination unit (U3) comprising a third pair of recombinase recognition sequences (RRS3) adapted to be recognized by the R1, R2, or a third recombinase (R3), wherein each of the RRS3 flanks each side of a third nucleic acid sequence, wherein the RRS3 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS3 is positioned downstream of at least one of the RRS2, whereby when the R1, R2, or R3 recognizes the RRS3, the third nucleic acid sequence is excised when the RRS3 are in the same orientation, or is inverted when the RRS3 are in the inverse orientation; wherein the first, second or third nucleic acid sequence comprises a target gene, and wherein presence or absence of at least one of the R1, R2, and R3 operatively links the promoter to at least one of the first, second or third nucleic acid sequence, thereby driving expression of the first, second or third nucleic acid sequence. 
     In some embodiments of the nucleic acid logic cassette, the R2 does not recognize the RRS1 or RRS3. Stated another way, the R2 is orthogonal to the R1 and R3. In some embodiments, the R3 does not recognize the RRS1 or RRS2. Stated another way, the R3 is orthogonal to the R1 or R2. 
     In some embodiments of the nucleic acid logic cassette, the cassette is adapted to function as a decoder (e.g., 2-input 4-output decoder, 3-input 8-output decoder), multi-input AND gate, full adder, full subtractor, or half adder-subtractor. 
     In another aspect, the invention relates to a switch operable in a mammalian immune cell. The switch can be used to control the expression of a gene or a set of genes of interest. For examples, the genes of interest include, but are not limited to, chimeric antigen receptor, T cell receptor, cytokines (e.g., IL-2, IL-12, IL-15), and suicide genes (e.g., HSV-TK, iCasp9). The switch comprises: (i) a nucleic acid sequence encoding a mammalian promoter; (ii) a first pair of recombinase recognition sequences (RRS1) for a first recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter; (iii) a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1; and (iv) a target gene positioned downstream of at least one of the RRS1, whereby expression of the target gene is controlled by the presence or absence of at least one of the R1 and R2. The switch can be used in a variety of applications such as cell-based immunotherapy. 
     In some embodiments of any one of the above aspects, the mammalian cell is a human cell. 
     In some embodiments of any one of the above aspects, the human cell is a B cell. 
     In some embodiments of any one of the above aspects, the human cell is a T cell. 
    
    
     
       DETAILED DESCRIPTION OF THE FIGURES 
         FIGS. 1A-1D  shows orthogonal site-specific tyrosine recombinases and serine integrases enable implementation of multi-input AND gates in mammalian cells.  FIG. 1A  shows that recombinases can perform simple buffer (BUF) logic operations, either by tyrosine recombinase-mediated excision (left) or serine integrase-mediated inversion (right).  FIG. 1B  shows recombinases are tested for their recombination efficiency and orthogonality on all BUF logic reporters.  FIG. 1C  shows recombinases are tested for heterospecific recombination sites.  FIG. 1D  shows an exemplary 6-input AND-gate that produces GFP when all inputs are present. M.F.I.=mean fluorescence intensity; a.u.=arbitrary units. 
         FIGS. 2A-2B  shows exemplary 2-input BLADE platform can produce four distinct output functions based on two inputs.  FIG. 2A  shows an embodiment of a 2-input BLADE template on one plasmid with a single transcriptional unit. This template contains four distinct regions of DNA (addresses) downstream of a promoter. Each address corresponds to an output function and is accessed or deleted via site-specific DNA recombination. Each address can be programmed from different configurations ranging from zero-inputs to Boolean functions. The first address (Z 00 ), which is the closest to the promoter, corresponds to a state where no recombinase is expressed (A=0, B=0). If the Z 00  address contains a protein coding sequence, then that gene will be expressed. Gene expression from the other addresses downstream of Z 00  will be blocked by the presence of Z 00  protein coding region. In the presence of recombinase A, which corresponds to state (A=1, B=0), addresses Z 00  and Z 01  will be removed, thus moving address Z 10  directly downstream of the promoter and allowing gene expression of address Z 10  only to occur. Similarly, when only recombinase B is present (A=0, B=1), addresses Z 00  and Z 10  are excised, allowing Z 01  to be moved directly downstream of the promoter. Finally, when both recombinases are expressed (A=1, B=1), addresses Z 00 , Z 01 , Z 10  are all excised, thus placing Z 11  downstream of the promoter unobstructed by the other addresses.  FIG. 2B  shows an exemplary 2-input BLADE template with tagBFP, EGFP, iRFP720, and mRuby2 as addresses Z 00 , Z 10 , Z 01 , and Z 11  respectively. a.u.=arbitrary units. 
         FIGS. 3A-3B  show that over one hundred robust gene circuits with up to two inputs and two outputs implemented using the 2-input BLADE template.  FIG. 3A  shows that to generate 2-input, 2-output circuits, a 2-input BLADE template can be configured with different combinations of output functions: zero-output (transcription termination sequence), one-output (GFP or mCherry) or two-output (GFP-T2A-mCherry).  FIG. 3B  shows a diverse library of &gt;100 gene circuits, each shown as an individual column with predicted truth table GFP/mCherry ON/OFF behavior (black=no output, green=GFP ON, red=mCherry ON) and corresponding experimental averaged single-cell results obtained from flow cytometry. Shown above is an expanded view of one of the logic gates made using this platform. M.F.I.=mean fluorescence intensity. 
         FIG. 4  shows a field-programmable storage and retrieval of logic and memory using a Boolean Logic Look-Up Table (LUT). The Boolean Logic LUT is a six-input-one-output genetic device that receives two data inputs, A and B, and is controlled by four select inputs, S 1 , S 2 , S 3 , and S 4 , producing an output of GFP. The select inputs are used to change data input-output behavior; each combination configures the device to any of the sixteen Boolean logic gates. F.I.=fluorescence intensity. 
         FIGS. 5A-5B  show that a 3-input BLADE template can be applied to create 3-input arithmetic computational circuits.  FIG. 5A  shows that an exemplary 3-input BLADE template can receive up to three inputs and has eight distinct outputs.  FIG. 5B  shows an exemplary three 3-input-2-output binary arithmetic computational circuits made using the 3-input BLADE template. The full adder can add A+B+C while the full subtractor calculates A−B−C. For addition, input C, output P, and output Q represent Carry In, Carry Out and Sum, respectively. For subtraction, input C, output P, and output Q signify Borrow In, Borrow Out and Difference, respectively. The half adder-subtractor performs either binary addition of A+B or binary subtraction of A−B depending on the presence of select input C. F.I.=fluorescence intensity. 
         FIGS. 6A-6C  shop recombinase cross-reactivity dose-response profile of Cre, Dre, VCre and Vika.  FIG. 6A  is a summary of intended and unintended recombination of Cre, Dre, VCre and Vika site-specific recombinases. Cre and Dre are mutually cross-reactive, whereas VCre can recombine Vika&#39;s recombination sites, but not the other way around.  FIG. 6B  shows a dose-response profile of Cre (left) and Dre (right) on both Cre and Dre reporter constructs.  FIG. 6C  shows a dose-response profile of VCre (left) and Vika (right) on both VCre and Vika reporter constructs. 
         FIG. 7  shows a recombinase-based 2-input, 1-output Boolean logic gates using Cre and Flp recombinases. Recombination sites for Cre and Flp are placed around termination sequences or GFP to enable or disable GFP expression. In this fashion all sixteen Boolean logic functions were created in mammalian cells. 
         FIGS. 8A-8B  show alternative versions of the Boolean Logic Look-up Table (LUT). ( FIG. 8A ) A Boolean Logic LUT using Dre, Vika, B3 and VCre as select inputs. ( FIG. 8B ) The same Boolean LUT using low amounts of select inputs to reduce cross-reactivity effects of Cre/Dre and VCre/Vika, as noticeably illustrated in the AND and A gates. 
         FIG. 9  shows that a unique Nucleotide Sequence guided assembly provides a fast and modular approach for creating DNA constructs. A library of 2-Input 2-Output circuit can be used by using a combinatorial Gibson assembly strategy using the 2-4 decoder framework as the basis, and 4 parts can be constructed for each slot—stop cassette, GFP, mCherry, and GFP-2A-mCherry. Since the 2-4 decoder has 4 slots, a total of 16 components can be used. Then a combinatorial assembly strategy can be employed to create all 256 possible 2-Input 2-Output circuits. Genes are first cloned into part entry vectors that contain 40 bp unique nucleotide sequences (UNSes). These part vectors are then digested with either Asc1+Not1 or Asc1+Nhe1 restriction endonucleases to expose UNSes. The part fragments are then gel purified and assembled into a linearized destination vector using Gibson isothermal assembly. 
         FIGS. 10A-10B  show the construction of BLADE constructs using Unique Nucleotide Sequence Guided Assembly.  FIG. 10A  shows that to create a 3:8 BLADE construct, part vectors are created that contain output states for each address. The part vectors, along with a destination (DEST) vector, are digested and gel purified to expose unique nucleotide sequences (UNSes).  FIG. 10B  shows the part fragments and linearized destination vector are then assembled together in order of UNS via Gibson isothermal assembly. 
         FIGS. 11A-11B  shows an exemplary buffer gate construct.  FIG. 11A  is a schematic of the construct that, when recombinase is expressed, it will bind to the two recombination sites and excise the stop cassette between the recombination sites. For serine recombinases, the recombination sites are placed between a GFP that is in antisense direction, so that the expression of the corresponding serine recombinase inverts the DNA fragment between the recombination sites, inverting the GFP into the sense direction and allowing GFP to be expressed.  FIG. 11B  shows that transient transfection of the recombinase and the reporter plasmid, most of the enzymes tested are highly active and sufficiently orthogonal for the circuit design. 
         FIGS. 12A-12C  show embodiments of the multi-Input AND gates which are a single layer and can be used in the mammalian genetic systems of the 16 2-Input Boolean logic gates shown in  FIG. 7 .  FIG. 12A  shows embodiments of different 2-input AND gates and resulting GFP expression with different input SSRs; bxb1, PhiC31, Cre, Flp, SCre, VCre, Vika, Dre, B3 and KD. Note that the AND is created by placing two stop cassettes in tandem. Because of the othoganoality of the recombinases, it becomes possible to generate multi-Input AND gates simply by placing more stop cassettes in tandem between a GFP and the promoter.  FIG. 12B  shows embodiments of the genetic construct circuits of exemplary 4-input, 6-input and 8-input AND gates with different input SSRs (othogonal recombinases); bxb1, PhiC31, Cre, Flp, SCre, VCre, Vika, Dre, B3 and KD.  FIG. 12C  shows GFP expression from the genetic construct circuits of shown in  FIG. 12B  with exemplary 4-input, 6-input and 8-input AND gates with different input SSRs; bxb1, PhiC31, Cre, Flp, SCre, VCre, Vika, Dre, B3 and KD. 
         FIGS. 13A-13D  shows exemplary logic gates, including a 2-to-4 decoder, a 3-input-2-output, a half adder, a half subtractor and a full adder gate.  FIG. 13A  shows an exemplary 2-to-4 decoder and a 3-input-2-output logic gate, demonstrating a signal to noise ratio of over 100 for each state for the 2-to-4 decoder.  FIG. 13B  shows exemplary half adder logic gates, using 1, 2 or 3 plasmids, with a stop cassette in the Z00 slot, a GFP in Z10 and Z01, and a GFP-2A-mCherry cassette in Z11.  FIG. 13C  shows an exemplary half substractor logic gate using 1, 2 or 3 plasmids where a stop cassette is placed in the Z00 slot, a GFP in Z01, and a GFP-P2A-mCherry in Z10. FIG.  13 D shows an exemplary full adder logic gate, a 3-input 2-output logic gate, using Cre, Flp and Vcre as input, where the slots are filled with either a stop cassette, GFP, mCherry or GFP-2A-mCherry, depending on the output of the full adder truth table, and resulting mCherry and GFP expression depending on the input shown in the truth table. 
         FIGS. 14A-14B  shows exemplary 2-to-4 decoders and 3-to-8 decoders using serine recombinases, which has a complementary directionality factor that when expressed together with the recombinase, will revert the inversion generated by the recombinase back to the original configuration.  FIG. 14A  shows an exemplary 2-to-4 decoder, using the inversion property of serine recombinases, and depending on the presence of directionality factor or not, which can revert the system back to the original state.  FIG. 14B  shows an exemplary 3-to-8 decoder, using the inversion property of serine recombinases, and depending on the presence of directionality factor or not, which can revert the system back to the original state. 
         FIG. 15A  shows a 2-input BP Advanced Excision and Integration Synthetic (AXIS) template on one plasmid with a single transcriptional unit. This template contains four distinct regions of DNA (addresses) downstream of a promoter. Each address corresponds to an output function and is accessed via site-specific DNA recombination. The first address (Z 00 ), which is the closest to the promoter, corresponds to a state where no recombinase is expressed (A=0, B=0). If the Z 00  address contains a protein coding sequence, then that gene will be expressed. Gene expression from the other addresses downstream of Z 00  will be blocked by the presence of Z 00  protein coding region. In the presence of recombinase A, which corresponds to state (A=1, B=0), each DNA cassette between recombinase A&#39;s two orthogonal pairs of attB/P recombination sites, will each be inverted, thus moving address Z 10  directly downstream of the promoter and allowing gene expression of address Z 10  only to occur. Similarly, when only recombinase B is present (A=0, B=1), the entire DNA cassette between the recombinase B&#39;s attB/P recombination sites will be inverted, allowing Z 01  to be moved directly downstream of the promoter. Finally, when both recombinases are expressed (A=1, B=1), DNA cassettes between both recombinase A and B&#39;s pairs of attB/P recombination sites will be inverted, thus placing Z 11  downstream of the promoter unobstructed by the other addresses. 
         FIG. 15B  shows a schematic of the 2-input BP AXIS template with iRFP720, tagBFP, mRuby2, and EGFP as addresses Z 00 , Z 10 , Z 01 , and Z 11  respectively. Also shown is averaged single-cell results of the 2-Input BP AXIS decoder using PhiC31 and TP901-1 integrases. Mean fluorescence intensity from n=3 independent transfections. a.u.=arbitrary units. 
         FIG. 16A  is a schematic illustrating the architecture of a full adder in accordance with some embodiments of the invention. “RRS for ” refers to the RRS in the forward orientation. “RRS rev ” refers to the RRS in the reverse orientation. “TG for ” refers to the TG in the forward orientation. “TG rev ” refers to the TG in the reverse orientation. “TT for ” refers to the transcriptional terminator in the forward orientation. R1 recognizes RRS1. R2 recognizes RRS2, RRS6, and RRS7. R3 recognizes RRS3, RRS4, and RRS5. 
         FIG. 16B  is a schematic illustrating the architecture of a full subtractor in accordance with some embodiments of the invention. R1 recognizes RRS1. R2 recognizes RRS2, RRS6, and RRS7. R3 recognizes RRS3, RRS4, and RRS5. 
         FIG. 16C  is a schematic illustrating the architecture of a half adder-subtractor in accordance with some embodiments of the invention. R1 recognizes RRS1. R2 recognizes RRS2, RRS6, and RRS7. R3 recognizes RRS3, RRS4, and RRS5. 
         FIG. 17A  is a schematic illustrating the architecture of a 2-input 4-output decoder in accordance with some embodiments of the invention. 
         FIG. 17B  is a schematic illustrating the architecture of a 2-input 4-output decoder in accordance with some embodiments of the invention. 
         FIG. 17C  is a schematic illustrating the architecture of a 2-input 3-output decoder in accordance with some embodiments of the invention. 
         FIG. 17D  is a schematic illustrating the architecture of a 3-input 8-output decoder in accordance with some embodiments of the invention. 
         FIG. 17E  is a schematic illustrating the architecture of a 3-input 8-output decoder in accordance with some embodiments of the invention. 
         FIG. 18  shows structure of switches in State 1 and State 2. Using the stable inversion switch, the expression of CARs can be controlled to control T-cell response to an antigen. 
         FIG. 19  shows a strategy to improve the flexibility and strength of adoptive T-cell therapy. Using dials that respond to the addition of a drug to tune the therapy to a patient&#39;s needs. 
         FIG. 20  shows stable inversion switching of a Cre/lox promoter switch controlling mCherry expression. 
         FIGS. 21A-21C  show tamoxifen induction in Jurkat T-cells of Cre activity on ( FIG. 21A ) a fluorescent reporter stable inversion switch. Seven days after induction, ( FIG. 21B ) mCherry expression decreases as the gene is excised and ( FIG. 21C ) GFP expression is turned on. 
         FIGS. 22A-22C  show drug-induced activation of Jurkat in response to the Her2 antigen using ( FIG. 22A ) an On switch that turns on expression of a Her2-specific CAR tagged to mCherry. ( FIG. 22B ) Expression of CAR increases upon induction and ( FIG. 22C ) Activation of the T-cell occurs in response to the antigen. 
         FIGS. 23A-23C  show the implementation of fluorescent stable inversion switch in primary T-cells. ( FIG. 23A ) Structure of the switch. Switching was observed for both the ( FIG. 23B ) Cre/lox and ( FIG. 23C ) Flp/frt system. 
         FIGS. 24A-24C  show that 2-input LR AXIS platform can produce four distinct outputs based on two integrase and RDF inputs. ( FIG. 24A ) 2-input LR AXIS template on one plasmid with a single transcriptional unit. This template contains four distinct regions of DNA (addresses) downstream of a promoter. Each address corresponds to an output function and is accessed via site-specific DNA recombination. The first address (Z 00 ), which is the closest to the promoter, corresponds to a state where no recombinase is expressed (A=0, B=0). If the Z 00  address contains a protein coding sequence, then that gene will be expressed. Gene expression from the other addresses downstream of Z 00  will be blocked by the presence of Z 00  protein coding region. In the presence of recombinase A and its associated RDF, which corresponds to state (A=1, B=0), each DNA cassette between recombinase A&#39;s two orthogonal pairs of attL/R recombination sites, will each be inverted, thus moving address Z 10  directly downstream of the promoter and allowing gene expression of address Z 10  only to occur. Similarly, when only recombinase B and its associated RDF are present (A=0, B=1), the entire DNA cassette between the recombinase B&#39;s attL/R recombination sites will be inverted, allowing Z 01  to be moved directly downstream of the promoter. Finally, when both recombinases and RDFs are expressed (A=1, B=1), DNA cassettes between both recombinase A and B&#39;s pairs of recombination attL/R sites will be inverted, thus placing Z 11  downstream of the promoter unobstructed by the other addresses. ( FIG. 24B ) A schematic of the 2-input LR AXIS template with iRFP720, tagBFP, mRuby2, and EGFP as addresses Z 00 , Z 10 , Z 01 , and Z 11  respectively ( FIG. 24C ) Averaged single-cell results of the 2-Input LR AXIS decoder using PhiC31 integrase and gp3, and TP901-1 integrase and orf7. Mean fluorescence intensity from n=3 independent transfections. a.u.=arbitrary units. 
         FIG. 25  shows the mechanism of recombinase-based stable inversion switch. The stable inversion switch is a two-step process that takes the cell from expressing one gene to another upon recombination. Using pairs of recombination sites, this switch allows for stable memory of the change in genetic expression. In addition, this switch can be used to control the promoter driving gene expression. 
         FIG. 26A  shows an order-dependent, five-color and five-state finite state machine. With no recombinase input, the circuit produces BFP. Upon addition of input A, Cre recombinase, acts on the circuit to yield GFP expression; if input B, Flp recombinase, is added next, the circuit yields mRuby2 expression. If input B is introduced first to the circuit, the output is iRFP rather than GFP and if input A is added next, expression of OFP is produced. 
         FIG. 26B  shows state table and state diagram representations of the five-color, five-state finite state machine. 
         FIG. 26C  shows a ripple carry adder circuit is created through connection of a half adder and full adder. The circuit can add two 2-bit inputs, thereby allowing summation up to 3+3=6. 
         FIG. 26D  shows a multiplier circuit is created through connection of four AND gates and two half adders. The circuit can multiply two 2-bit inputs, thereby allowing multiplication up to 3*3=9. 
         FIG. 26E  shows an arithmetic logic unit that permits all sixteen basic boolean logic functions as well as performing as a half adder. 
         FIG. 26F  shows an arithmetic logic unit that permits all sixteen basic boolean logic functions as well as performing as a half adder or a half subtractor. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a platform to construct complex nucleic acid logic cassettes on single transcriptional units. The nucleic acid logic cassettes can perform simple or complex logic functions, e.g., in mammalian cells, including, but not limited to, A, N, NOT A, NOT B, NOR, OR, AND, NAND, A IMPLY B, B IMPLY A, A NIMPLY B, B NIMPLY A, XOR, XNOR, decoder, half adder, half subtractor, half adder-subtractor, full adder, full subtractor, Feynman gate, logic selector, memory, Ripple carry adder, array multiplier, arithmetic logic unit, and any combinations thereof. To perform a logic function, a nucleic acid logic cassette can receive at least one input, preferably at least two inputs (e.g., 2, 3, 4, 5, 6, 7, or more), and produce at least one output (e.g., 1, 2, 3, 4, 5, 6, or more) as a function of the input(s). 
     To perform complex logic functions, existing genetic logic circuits assemble a complex circuitry from a plurality of simple cassettes; signal relay is required as an output from one cassette serves as an input for another cassette. Given the complexity of the assembly, such multi-layered approach makes these logic circuits difficult to reproduce and insert into cells. In addition, at least due to the requirement for signal relay, the fidelity of these logic circuits is low. And thus using these logic circuits in higher organisms such as mammals has been challenging. In contrast, because the logic cassettes of the present invention are constructed on single transcriptional units, no signal relay is necessary, and they are easy to reproduce and insert into cells. The complexity lies in the design of the logic cassettes on the back end, which ultimately allows simple and reliable utilization of these logic cassettes on the front end. Due to these significant advantages, the logic cassettes described herein have been demonstrated by the inventors to work in mammalian cells such as human cells. 
     The methodology of constructing complex nucleic acid logic cassettes takes advantage of the simple operation principles of site-specific recombinases. As shown in  FIG. 11A , when a nucleic acid sequence (e.g., a target gene) is flanked by a pair of recombinase recognition sequences (RRS) in the same orientation, the nucleic acid sequence can be excised upon the recognition of the RRS by the proper recombinase; when a nucleic acid sequence is flanked by a pair of recombinase recognition sequences (RRS) in an inverse orientation, the nucleic acid sequence can be inverted upon the recognition of the RRS by the proper recombinase. The inversion and/or excision reactions will place the nucleic acid sequence (e.g., a target gene) in a new location and/or orientation to make it operatively linked to a promoter, thereby driving expression of the nucleic acid sequence. Based on these operation principles, depending on the combination of excision and inversion of 1, 2, or more nucleic acid sequence(s), one can select from the growing number of recombinases and their corresponding RRS to construct nucleic acid logic cassettes for a specific logic function. 
     The construction of the nucleic acid logic cassettes can be modular. Each module can be a nucleic acid sequence flanked by a pair of RRS. Depending on the specific logic functions, the modules can be connected in series, or there can be overlapping nucleic acid regions between the modules. A truth table for a desired logic function can be used to guide the placement of each module. Generally, a truth table is composed of one column for each input variable (for example, A and B), and one final column for all of the possible results of the logical operation that the table is meant to represent (for example, A XOR B). 
     It has been reported in US2014/0315310, which is incorporated herein by reference, the creation of recombinase-based logic gates in bacterial cells only. US2014/0315310 describes simple recombinase-based logic gates, i.e., logic gates that require no more than two inputs and produce no more than one output. In contrast, the present invention relates to recombinase-based expression vectors or gene circuits in mammalian cells. For example, the inventors have used recombinases and their corresponding heterospecific DNA binding sites to create all sixteen Boolean logic gates in the human embryonic kidney cell line. Additionally, the inventors have created expression vectors that can perform complex logic functions with more than two inputs (e.g., 3, 4, 5, 6, 7, 8, or more) and/or more than one output (e.g., 2, 3, 4, or more) in a single transcription unit. This is a significant advantage, as the present invention provides methods and cassettes for complex logic functions in mammalian cells using a series arrangement of simple modules in a single cassette. 
     The nucleic acid logic cassettes described herein can be used in vitro or in vivo. An example of in vitro use is the study of cell culture, in which cells contain the nucleic acid logic cassettes described herein. An example of in vivo use is the use of the nucleic acid logic cassettes described herein to regulate or control gene expression in a subject such as a human. 
     Genetic Circuit Platforms 
     Through the use of site-specific DNA recombinases (SSR) and their corresponding heterospecific DNA binding sites (e.g., recombinase recognition sequences (RRS)), the inventors have constructed and demonstrated, in mammalian cells, a suite of multi-input-multi-output (MIMO) circuits, such as a Half Adder, Half Subtractor, 2-Input Decoder, Full Adder, Full Subtractor, and Half Adder-Subtractor. Moreover, the inventors also created a Programmable Read Only Memory (PROM) device that can select between 16 2-Input logic gates based on 4 inputs. More importantly, the inventors were able to create all of the computation circuits in a single transcription unit (e.g., cassette)—no linkages between different transcription units are required to achieve the desired circuits. This development bypasses one of the most difficult challenges in synthetic circuit design, and allows all of the circuits to be successfully implemented on first attempt without optimization. For example, it is not necessary to have multiple plasmids for each logic function, nor is it necessary to calibrate the ratios of each plasmid with respect to each other for the correct logic function. This platform provides a powerful tool for complex gene regulation and expression in mammalian cells and high-level mammalian cells reprogramming for development of animal models. Furthermore, the circuit design platform described herein minimizes the typical “design-test-build” cycle that plagues the progress of most synthetic biology projects, and illustrates a new paradigm in the design of synthetic genetic logic circuits. 
     The inventors have demonstrated the use of the platform for the generation of N-input order-independent logic gates called Boolean Logic and Arithmetic through DNA Excision (BLADE). All logic computation for BLADE circuits is done on a single transcriptional layer and circuit construction is based on a BLADE template. N-input BLADE templates feature 2 N  distinct regions of DNA termed addresses (Z). Each address is accessible only via a specific combination of inputs. For example, the 2-input BLADE template contains four addresses, Z=Z AB =Z 00 , Z 10 , Z 01 , and Z 11 , corresponding to each state of A and B inputs (see  FIG. 2A ). Each address contains one output function, which ranges from having zero outputs (e.g., transcription terminator), through arbitrary numbers of outputs separated by ribosomal skip sequences (2A), to Boolean functions like buffer (BUF). A 2-input 4-output decoder has been constructed using the BLADE platform (e.g.,  FIG. 2B ). 
     Extending the BLADE framework further, the inventors also demonstrate a 3-input BLADE template (in some embodiments as a 3-input 8-output decoder) for constructing sophisticated arithmetic functions in human cells ( FIG. 5A ). Such a 3-input BLADE genetic circuit responds to three inputs (Cre, Flp, and VCre) and contains eight addresses for expression of up to eight distinct output functions. This design utilizes three different heterospecific sites (e.g., RRS) for Cre and Flp, but just one site for VCre. Three 3-input-2-ouput arithmetic computational circuits were made and tested in mammalian cells from the 3-input BLADE template ( FIG. 5B ). The full adder and full subtractor can perform either binary addition or subtraction of three 1-bit inputs, respectively. Furthermore, the half adder-subtractor is an arithmetic FPROM circuit that can compute addition or subtraction on two data inputs, A and B, depending on the presence of one select input C. 
     Similarly, exploiting the principal of Field-Programmable Read-Only Memory (FPROM) in electronic circuits, the inventors configured the input-output behavior of these circuits in the field post-manufacturing, allowing them to customize their circuits and devices at a later time. In particular, the inventors built a FPROM device termed a Boolean Logic Look-Up Table (LUT) that is based on placing BUF gates into the four addresses of the 2-input BLADE template (see  FIG. 4 ). This circuit has two data inputs, A and B, and four select inputs, S 1 , S 2 , S 3 , and S 4 . Each select input can control which buffer gates are GFP ON or OFF. Thus, each combination of select inputs configures the device to a specific Boolean logic gate with up to two inputs and one output. For instance, an OR function can be achieved using select inputs S 2 , S 3 , and S 4 , keeping address Z 00  GFP OFF and setting addresses Z 10 , Z 01 , and Z 11  GFP ON. This circuit behaves as expected in mammalian cells. 
     Accordingly, the present invention provides a platform which is the foundation for generating logic gates with arbitrary number of inputs and outputs. 
     The following are examples of the use of the platform:
         1. As powerful genetic tools for investigating and manipulating genetic functions
           Cell-type expression of exogenous genes.
               Expression of optogenetic proteins for optical control and study of specific cell types in the brain   Delivery of proteins/peptides/RNAs for therapeutic purposes   
               Cell-type overexpression, knockdown, knockout or mutation of endogenous genes (via genome modifying proteins such as TAL, Zinc finger and Cas9 effectors and nucleases)
               Generation of animal models for study of diseases (e.g. cancer, neurological diseases, and other genetic pathologies)   Therapeutic correction of a pathogenic gene (e.g. repression of a cancer-causing oncogene or reactivation of a mutated tumor suppressing gene)   
               
           2. As advanced implantable cellular computers for seek-and-destroy or diagnostic purposes
           a. Cell-based therapeutics
               i. Cancer cell and pathogen seek-and-destroy engineered immune cells   
               b. Cell-based diagnostics
               i. Disease biomarker sensing (e.g. detection of overexpression of an oncogene)   ii. Metabolite sensing and regulation (e.g. blood sugar regulation)   iii. Pathogen detection (e.g. HIV, pathogenic bacteria)   
               
               

     The biological devices and circuits as disclosed herein have wide-reaching applications and commercial interest. As a research tool, this technology can allow a scientist to genetically target specific cell-types in the body for studying and manipulating physiological functions. Moreover, cell-based gene therapies have shown tremendous clinical success and investor interest. The technology described herein can vastly enhance such strategies by integrating advanced cellular computations. For instance, one can create implantable cellular computers that could act as early-warning systems for diseases, attack cancerous cells, or monitor and regulate blood-glucose levels. Current technologies can&#39;t integrate the large numbers and logical nature of biological and environmental signals that these applications would require. 
     Many commercial applications would also benefit by the use of the genetic logic gates as disclosed herein. For example, adoptive T cell therapy is a type of gene therapy that has had tremendous clinical success and gained significant investor interest. The therapy involves the replacement of a cancer patient&#39;s immune cells with genetically engineered ones that are programmed with a chimeric antigen receptor (CAR) that targets proteins overexpressed on cancer cells. However, most other cancers are not as easily targeted since markers are not exclusively expressed on the surface of the cells. The use of the genetic logic gates as disclosed herein can offer a novel solution for these difficulties to target cancers by employing digital logic. 
     With the genetic logic gate systems disclosed herein, one is not limited to just targeting proteins that are overexpressed on the surface of cancer cells, it can be used to also induce or repress activation of the genetically engineered T cells with the abundance or absence of cellular and environmental signals. For instance, it can be used to limit off-target effects by limiting activation of the engineered T cells to the location of the cancer. This can be done via logical integration of multiple signals, such as the presence/absence of metabolites and cytokines found in the cancer, oxygen levels (i.e. tumor hypoxia) and spatial release of a chemical inducer. 
     Others have utilized other strategies for programming digital logic in living organisms. Most utilize transcription-factor based methods in both bacteria and in mammalian cells. DNA-binding proteins are expressed that either promote or repress gene expression on the transcriptional level. Although this method can be highly modular, a plurality levels of cascading components is required to achieve complex logical tasks. A plurality of plasmids is often necessary, which makes implementation into organisms more difficult, as all of the elements need to be expressed in single cells and at the correct ratio for these systems to function properly. 
     In contrast, the genetic logic gates as disclosed herein are advantageous in that they rely on the use of SSR and their corresponding RRS. Accordingly, one can create a single nucleic acid cassette capable of performing complicated logical tasks by utilizing heterospecific recombination sites. For instance, one of the genetic circuits, the full adder, is a three-input, two-output logic circuit that can digitally add up to three. It requires only one plasmid, far fewer than transcription-factor-based strategies would necessitate, thus making incorporation of all of the components into the genome of a single cell much easier. Therefore, the platform as disclosed herein using site-specific recombinases is well suited for the wide variety of commercial applications involving the need for cell-type specification and sensing of environmental signals. 
     In one aspect, the invention relates to a nucleic acid logic cassette comprising: (i) a nucleic acid sequence encoding a promoter; (ii) a first recombination unit (U1) comprising a first pair of recombinase recognition sequences (RRS1) for a first site-specific recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter, whereby when the R1 recognizes the RRS1, the first nucleic acid sequence is excised when the RRS1 are in the same orientation or is inverted when the RRS1 are in the inverse orientation; (iii) a second recombination unit (U2) comprising a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1, whereby when the R2 recognizes the RRS2, the second nucleic acid sequence is excised when the RRS2 are in the same orientation or is inverted when the RRS2 are in the inverse orientation; (iv) a third recombination unit (U3) comprising a third pair of recombinase recognition sequences (RRS3) adapted to be recognized by the R1, R2, or a third recombinase (R3), wherein each of the RRS3 flanks each side of a third nucleic acid sequence, wherein the RRS3 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS3 is positioned downstream of at least one of the RRS2, whereby when the R1, R2, or R3 recognizes the RRS3, the third nucleic acid sequence is excised when the RRS3 are in the same orientation, or is inverted when the RRS3 are in the inverse orientation; wherein the first, second or third nucleic acid sequence comprises a target gene, and wherein presence or absence of at least one of the R1, R2, and R3 operatively links the promoter to at least one of the first, second or third nucleic acid sequence, thereby driving expression of the first, second or third nucleic acid sequence. 
     In some embodiments, the R2 does not recognize the RRS1 or RRS3. Stated another way, the R2 is orthogonal to the R1 and R3. In some embodiments, the R3 does not recognize the RRS1 or RRS2. Stated another way, the R3 is orthogonal to the R1 or R2. 
     In some embodiments, the nucleic acid logic cassette further comprises a pair of flanking nucleic acid sequences, wherein each of the flanking nucleic acid sequences flanks each side of the nucleic acid logic cassette, permitting the cassette to be inserted into the genome of a mammalian cell. Examples of flanking nucleic acid sequences include, but are not limited to, AAVS1 and Rosa26 left and right arms. 
     In some embodiments, the U1, U2, and U3 can be connected in series. In some embodiments, there can be an overlap between the U1 and U2, U1 and U3, or U2 and U3. In some embodiments, the U1 can comprise the U2. In some embodiments, the U1 can comprise the U3. In some embodiments, the U2 can comprise the U3. In some embodiments, the U1 can comprise the U2 and U3. 
     In some embodiments, the nucleic acid logic cassette can further comprise a fourth recombination unit (U4) comprising a fourth pair of recombinase recognition sequences (RRS4) adapted to be recognized by the R1, R2, R3, or a fourth recombinase (R4), wherein each of the RRS4 flanks each side of a fourth nucleic acid sequence, and wherein the RRS4 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS4 is positioned downstream of at least one of the RRS3, whereby when the R1, R2, R3, or R4 recognizes the RRS4, the fourth nucleic acid sequence is excised when the RRS4 are in the same orientation or is inverted when the RRS4 are in the inverse orientation. 
     Factors that can be taken into consideration for designing the nucleic acid logic cassette include, but are not limited to, the number of recombination units, the position of each recombination unit, the orientation of each pair of RRS, the identity of each pair of RRS, the orientation of the genetic elements (e.g., promoters, transcriptional terminators, target genes), the number of genetic elements, and the type of genetic elements. 
     In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is an embryonic stem cell. In some embodiments, the mammalian cell is an immune cell such as T or B cell. It should be noted that the technology described herein can also be applied to non-mammalian cells such as bacteria. 
     In some embodiments, the cassettes described herein can perform logic functions with memory, where removal of the input afterwards does not make the circuit return to its original state. 
     In some embodiments, the cassettes described herein can perform logic functions without memory, where removal of the input afterwards returns the circuit to its original state. In some of these embodiments, serine integrases can be used. Integrases (which do not recognize the attL/R sites on their own) and the recombination directionality factors (RDFs) are the main sensing inputs that convert the L/R sites to B/P. However, upon removal of the RDFs, the circuit returns to its original state due to the constitutively expressing integrases that convert the B/P sites back to L/R. 
     In some embodiments, a plurality of nucleic acid logic cassettes can be assembled to carry out a desired logic function. 
     In one aspect, the invention relates to a logic system comprising a nucleic acid logic cassette described herein, and at least one nucleic acid sequence encoding at least one inducible promoter operatively linked to at least one recombinase, whereby expression of the at least one recombinase provides an input to the nucleic acid logic cassette adapted to perform a logic function as a function of the input. 
     In some embodiments, the logic system comprises two or more nucleic acid sequences, wherein each of the nucleic acid sequences can encode an inducible promoter and a recombinase operatively linked to the promoter. 
     In some embodiments, a single nucleic acid sequence can encode two or more recombinases operatively linked to an inducible promoter. 
     In some embodiments, the logic system can further comprising at least one nucleic acid sequence encoding at least one inducible promoter operatively linked to a RDF specific for the at least one recombinase. 
     In one aspect, the invention relates to a mammalian cell containing a nucleic acid logic cassette or logic system described herein. The logic cassette or system can be introduced into a cell using any method known to one of skill in the art. The term “transformation” as used herein refers to the introduction of genetic material into a cell, tissue or organism. Transformation of a cell can be stable or transient. The term “transient transformation” or “transiently transformed” refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell&#39;s genome. Transient transformation can be detected by, for example, enzyme linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by one or more of the transgenes. For example, a molecular circuit can further comprise a promoter operatively linked to an output product, such as a reporter protein. Expression of that reporter protein indicates that a cell has been transformed or transfected with the molecular circuit, and is hence implementing the circuit. Alternatively, transient transformation can be detected by detecting the activity of the protein encoded by the transgene. The term “transient transformant” refers to a cell which has transiently incorporated one or more transgenes. 
     In contrast, the term “stable transformation” or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell or cellular system, preferably resulting in chromosomal integration and stable heritability through meiosis. Stable transformation of a cell can be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes. Alternatively, stable transformation of a cell can also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences. The term “stable transformant” refers to a cell or cellular, which has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene. Transformation also includes introduction of genetic material into plant cells in the form of plant viral vectors involving epichromosomal replication and gene expression, which can exhibit variable properties with respect to meiotic stability. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. 
     In one aspect, the invention relates to a mammalian cell containing a nucleic acid logic cassette, the cassette comprising: (i) a nucleic acid sequence encoding a mammalian promoter; (ii) a first recombination unit (U1) comprising a first pair of recombinase recognition sequences (RRS1) for a first recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter, whereby when the R1 recognizes the RRS1, the first nucleic acid sequence is excised when the RRS1 are in the same orientation or is inverted when the RRS1 are in the inverse orientation; (iii) a second recombination unit (U2) comprising a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1, whereby when the R2 recognizes the RRS2, the second nucleic acid sequence is excised when the RRS2 are in the same orientation or is inverted when the RRS2 are in the inverse orientation, wherein the first or second nucleic acid sequence comprises a target gene, and wherein presence or absence of at least one of the R1 and R2 operatively links the promoter to at least one of the first and second nucleic acid sequence, thereby driving expression of the first or second nucleic acid sequence. In some embodiments, the mammalian cell is an immune cell. In some embodiments, the cassette can perform a logic function selected from the group consisting of NOR, OR, AND, NAND, A IMPLY B, B IMPLY A, A NIMPLY B, B NIMPLY A, XOR, XNOR, decoder, half adder, half subtractor, half adder-subtractor, full adder, full subtractor, Feynman gate, logic selector, Ripple carry adder, array multiplier, arithmetic logic unit, and any combinations thereof. 
     Switches for Adoptive T-Cell Therapy 
     One aspect of the invention relates to nucleic acid-based switches and their use in adoptive T-cell therapy. These nucleic acid-based switches are based on the DNA recombinase systems. Chimeric antigen receptors (CARs) direct T-cell activity towards cancer cells. CARs are a fusion of the single chain variable fragment (scFv) of an antibody fused to the signaling domain from the T-cell receptor (TCR). 
     In one aspect, the invention relates to a switch operable in a mammalian immune cell, comprising: (i) a nucleic acid sequence encoding a mammalian promoter; (ii) a first pair of recombinase recognition sequences (RRS1) for a first recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter; (iii) a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1; and (iv) a target gene positioned downstream of at least one of the RRS1, whereby expression of the target gene is controlled by the presence or absence of at least one of the R1 and R2. 
     The working mechanism of the switches described herein is a two-step process. In the first step, the presence of the R2 inverts a second nucleic acid sequence. This inversion is not stable as the switch keep switching uncontrollably. In the second step, the presence of the R1 excises a portion of the switch, thus making the inversion permanent. In some embodiments, the switching mechanism is shown in  FIG. 25 . 
     In some embodiments, the R1 and R2 are each a tyrosine recombinase. Without wishing to be bound by theory, one pair of tyrosine recombinase sites would lead to bidirectional inversion, i.e. the switch would keep switching uncontrollably. 
     In some embodiments, the R1 and R2 are each a serine recombinase. 
     The switches described herein can control the response of the T-cell to a target antigen.  FIG. 18  illustrates some embodiments of these switches. In some embodiments, the switch is a temporal switch. The temporal switch can be an On switch, which allows the cell to stay in an “off” state where no CAR is expressed until the drug is added, at which point the CAR is expressed and the T-cell is “on.” This design allows for a doctor to turn the CAR on only when they feel it is necessary. The temporal switch can also be an OFF switch, which begins with the T-cell in the “on” state, and upon drug addition, will turn the T-cell “off.” This switch can be used to turn the therapy off upon remission to minimize the possibility of cytotoxic effects. 
     In some embodiments of the On switch, there is an overlap between the first nucleic acid sequence and the second nucleic acid sequence. Specifically, the first nucleic acid sequence comprises one of the RRS2 and the target gene in an inverted orientation with respect to the promoter, and the second nucleic acid sequence comprises the target gene and one of the RRS1. The target gene is not expressed in the absence of the correct input. In the presence of the R2 and then R1, the target gene is inverted, thereby turning on the expression of the target gene. 
     In some embodiments of the Off switch, there is an overlap between the first nucleic acid sequence and the second nucleic acid sequence. Specifically, the first nucleic acid sequence comprises one of the RRS2 and the target gene in the same orientation as the promoter, and the second nucleic acid sequence comprises the target gene and one of the RRS1. The target gene is expressed in the absence of the correct input. In the presence of the R2 and then R1, the target gene is inverted, thereby turning off the expression of the target gene. 
     In some embodiments, the switch is a target switch. Different targets for CARs can provoke different responses in the T-cell due to the variability in antigen expression level and activation levels. As cancers can have multiple targets, this switch will allow for tuning of the therapy by changing the target. This switch can be adapted for different targets. 
     In some embodiments of the target switch, there is an overlap between the first nucleic acid sequence and the second nucleic acid sequence. Specifically, the first nucleic acid sequence comprises a first target gene in the same orientation as the promoter, one of the RRS2, and a second target gene in an inverted orientation. The second nucleic acid sequence comprises the second target gene and one of the RRS1. The first target gene is expressed in the absence of the correct input. In the presence of the R1 and R2, the second target gene is operatively linked to the promoter, thereby turning on the expression of the second target gene and turning off the expression of the first target gene. 
     In some embodiments, the switch is an affinity switch. T-cell activation using CARs is dependent in part on the affinity of the scFv for the target antigen. Anti-Her2 CARs have varying affinities and can be used to construct a switch that can change the affinity of the T-cell for the antigen. Her2 is an EGFR family receptor that is overexpressed in a number of cancers, including breast, colon, and ovarian cancer. Existing versions of Her2-CAR have affinities that span four orders of magnitude. In some embodiments, this switch will start with expression of a low-affinity CAR. While using a low-affinity CAR may reduce the probability of binding to a cancer cell, it will also reduce the probability of 
     binding to a healthy cell that expresses Her2. In the case that the patient does not respond to a low-affinity Her2-CAR, they will then be switched to a high-affinity Her2-CAR. 
     In some embodiments, the genetic architecture of the affinity switch is the same as that of the target switch. In these embodiments, the first target gene is a low-affinity CAR, and the second target gene is a high-affinity CAR. In some embodiments, the high-affinity CAR has an affinity to a target at least 20% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 3 fold) higher than that of the low affinity CAR to the same target. 
     In some embodiments, the switch is an expression level switch. Another method to impact T-cell activation is to alter the level of expression of CARs. One strategy to affect expression level is to change the promoter under which the CAR is expressed. In the expression level switch, the T-cell will start with the CAR transcribed under a low-expressing promoter. To increase the strength of the treatment, the circuit will then drive the expression of CAR under a different, high-expressing promoter. 
     In some embodiments of the expression level switch, the promoter is in an inverted orientation. There is an overlap between the first nucleic acid sequence and the second nucleic acid sequence. Specifically, the first nucleic acid sequence comprises the promoter and one of the RRS2. The second nucleic acid sequence comprises the promoter and one of the RRS1. The target gene is positioned downstream of the RRS1 and RRS2, whereby in the presence of the R1 and R2, the promoter is inverted, thereby turning on the expression of the target gene. In the absence of the correct input, the target gene is not expressed. 
     The switches described herein can be used in adoptive T-cell therapy. Adoptive T-cell therapy uses a patient&#39;s own immune cells to target and kill cancer cells. One of the primary challenges are potential autoimmune effects of the therapy, as many of the target antigens that are overexpressed on cancer cells can also be found at lower levels on healthy cells. While the use of CARs in some patients allows for targeted killing of cancer cells, other patients who express high amounts of the antigen on normal cells have experienced fatal autoimmune effects upon targeting of healthy tissue. By implementing the switches in the adoptive T-cell therapy, the autoimmune effects can be reduced. 
     Accordingly, in one aspect, a method is provided herein to treat cancer in a subject, the method comprising incorporating a switch described herein into T cells and administering the T cells to the subject. In some embodiments, the method further comprises administering a lymphodepletion procedure to the subject prior to the administration of the T cells. In a lymphodepletion procedure, drugs such as fludarabine and Cytoxan can be used to significantly reduce the number of normal lymphocytes circulating in the patient&#39;s body. 
     In some embodiments, the method further comprises culturing and/or expanding the T cells containing the switch prior to their administration. Methods of culturing and/or expanding cells are known in the art. For switches that comprise an inducible promoter, the method further comprises administering a compound to activate the switch. The compound can be selected from the group consisting of doxycycline, tamoxifen, rapamycin, and abscisic acid, depending on the inducible promoter. 
     Site Specific Recombinases (SSR) and Recombination Recognition Sequences 
     Provided herein are recombinases used to impart stable, DNA-base memory to the logic and memory systems of the invention. A “recombinase,” as used herein, is a site-specific enzyme that recognizes short DNA sequence(s), which sequence(s) are typically between about 30 base pairs (bp) and 40 bp, and that mediates the recombination between these recombinase recognition sequences, which results in the excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. A “genetic element,” as used herein, refers to a sequence of DNA that has a role in gene expression. For example, a promoter, a transcriptional terminator, and a nucleic acid encoding a product (e.g., a protein product) is each considered to be a genetic element. 
     Exemplary recombinases include, but are not limited to, Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, λ, HK022, HP1, γδ, ParA, Tn3, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, PhiMR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, and HbiF. 
     Exemplary RRS include, but are not limited to, loxP, loxN, lox511, lox5171, lox2272, M2, M3, M7, M11, lox71, lox66, FRT, rox, SloxM1, VloxP, vox, B3RT, KDRT, F3, F14, attB/P, F5, F13, Vlox2272, Slox2272, SloxP, RSRT, and B2RT. 
     Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases), based on distinct biochemical properties. Serine recombinases and tyrosine recombinases are further divided into bidirectional recombinases and unidirectional recombinases. Examples of bidirectional serine recombinases include, without limitation, β-six, CinH, ParA and γδ; and examples of unidirectional serine recombinases include, without limitation, Bxb1, ΦC31 (phiC31), TP901, TGI, φBTI, R4, cpRV1, cpFC1, MRU, A118, U153 and gp29. Examples of bidirectional tyrosine recombinases include, without limitation, Cre, FLP, and R; and unidirectional tyrosine recombinases include, without limitation, Lambda, HK1O1, HK022 and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange. Recombinases have been used for numerous standard biological applications, including the creation of gene knockouts and the solving of sorting problems. 
     In some embodiments, the recombinases for use in the present invention are orthogonal recombinases. When a first recombinase is orthogonal to the second recombinase, it means that the second recombinase does not recognize the RRS specific for the first recombinase, neither does the first recombinase recognize the RRS specific for the second recombinase. 
     The outcome of recombination depends, in part, on the location and orientation of two short repeated DNA sequences (e.g., RRS) that are to be recombined, typically less than 30 bp long. The site-specific recombinases bind to these repeated sequences, which are specific to each recombinase, and are herein referred to as “recombinase recognition sequences” or “recombinase recognition sites.” Thus, as used herein, a recombinase is “specific for” a recombinase recognition site when the recombinase can mediate inversion or excision between the repeat DNA sequences. As used herein, a recombinase may also be said to recognize its “cognate recombinase recognition sites,” which flank an intervening genetic element (e.g., promoter, terminator, or target gene). A genetic element is said to be “flanked” by recombinase recognition sites when the element is located between and immediately adjacent to two repeated DNA sequences. In some embodiments, the recombinase recognition sites do not overlap each other. However, in other embodiments, recombinase recognition sites do overlap each other, such as described herein below, which permits greatly increased combinatorial complexity. 
     Inversion recombination happens between two short, inverted, repeated DNA sequences. Without wishing to be bound by theory, a DNA loop formation, assisted by DNA bending proteins, brings the two repeat sequences together, at which point DNA cleavage and ligation occur. This reaction is ATP independent and requires supercoiled DNA. The end result of such an inversion recombination event is that the stretch of DNA between the repeated site inverts (i.e., the stretch of DNA reverses orientation) such that what was the coding strand is now the non-coding strand and vice versa. In such reactions, the DNA is conserved with no net gain or no loss of DNA. 
     Conversely, excision (integration) recombination occurs between two short, repeated DNA sequences that are oriented in the same direction. In this case, the intervening DNA is excised/removed. For example, an AND gate can be assembled by placing a terminator between each of two different sets of recombinase sites oriented for excision, flanked by a promoter and an output such as a GFP-encoding sequence. In this example, both terminators must be excised by input-dependent action of the recombinase(s) to permit readthrough from the promoter to the GFP-encoding sequence. Thus two inputs are needed to excise both terminators to generate output. 
     Recombinases can also be classified as irreversible or reversible. As used herein, an “irreversible recombinase” refers to a recombinase that can catalyze recombination between two complementary recombination sites, but cannot catalyze recombination between the hybrid sites that are formed by this recombination without the assistance of an additional factor. Thus, an “irreversible recognition site” refers to a recombinase recognition site that can serve as the first of two DNA recognition sequences for an irreversible recombinase and that is modified to a hybrid recognition site following recombination at that site. A “complementary irreversible recognition site” refers to a recombinase recognition site that can serve as the second of two DNA recognition sequences for an irreversible recombinase and that is modified to a hybrid recombination site following homologous recombination at that site. For example, attB and attP, described below, are the irreversible recombination sites for Bxb1 and phiC31 recombinases—attB is the complementary irreversible recombination site of attP, and vice versa. Recently, it was shown that the attB/attP sites can be mutated to create orthogonal B/P pairs that only interact with each other but not the other mutants [72]. This allows a single recombinase to control the excision or integration or inversion of multiple orthogonal B/P pairs. 
     The phiC31 (ϕC31) integrase, for example, catalyzes only the attB×attP reaction in the absence of an additional factor not found in eukaryotic cells. The recombinase cannot mediate recombination between the attL and attR hybrid recombination sites that are formed upon recombination between attB and attP. Because recombinases such as the phiC31 integrase cannot alone catalyze the reverse reaction, the phiC31 attB×attP recombination is stable. 
     Irreversible recombinases, and nucleic acids that encode the irreversible recombinases, are described in the art and can be obtained using routine methods. Examples of irreversible recombinases include, without limitation, phiC31 (ϕC31) recombinase, coliphage P4 recombinase, coliphage lambda integrase, Listeria A118 phage recombinase, and actinophage R4 Sre recombinase, HK101, HK022, pSAM2, Bxb1, TP901, TGI, ϕBTI, cpRV1, cpFC1, MRU, U153 and gp29. Conversely, a “reversible recombinase” refers to a recombinase that can catalyze recombination between two complementary recombinase recognition sites and, without the assistance of an additional factor, can catalyze recombination between the sites that are formed by the initial recombination event, thereby reversing it. The product-sites generated by recombination are themselves substrates for subsequent recombination. Examples of reversible recombinase systems include, without limitation, the Cre-lox and the Flp-frt systems, R, β-six, CinH, ParA and γδ. 
     The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the invention. The complexity of cassettes and systems of the invention can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities. Other examples of recombinases that are useful in the invention described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the invention. 
     In some embodiments, the recombinase is serine recombinase. Thus, in some embodiments, the recombinase is considered to be irreversible. For some serine recombinases, an initial recombination event can be reversed when a recombinase directionality factor (RDF) is present. RDFs are a diverse group of proteins involved in controlling the directionality of integrase-mediated site-specific recombination reactions. Typically, RDFs are small DNA-binding proteins acting as accessory factors to influence the choice of substrates that are recombined by their cognate recombinase. See Lewis and Hatfull, Nucleic Acids Res. 2001 Jun. 1; 29(11): 2205-2216. For example, when the recombination sites, attB and attP are placed in the antiparallel orientation, the presence of recombinases Till stably invert the DNA sequence between the two sites and generate an attL and attR site (“BP reaction”). This inversion remains stable unless a RDF is also expressed along with bxb1 or phiC, which will invert the sequence between attL and attR and regenerate attB and attP site (“LR reaction”). Examples of RDF include, but are not limited to, gp47 for bxb1, gp3 for phiC31, gp3 for PhiBT1, ORF7 for TP901-1, gp25 for TG1, and gp3 for PhiRv1. 
     In some embodiments, the recombinase is a tyrosine recombinase. Thus, in some embodiments, the recombinase is considered to be reversible. 
     In some embodiments, all the recombinases for a specific nucleic acid logic cassette can be of the same type (e.g., serine or tyrosine). For example, the BLADE platform described herein utilizes a plurality of tyrosine recombinases; the AXIS platform described herein utilized a plurality of serine recombinases. In some embodiments, tyrosine recombinases and serine recombinases can be used together in the same logic cassette. 
     In some embodiments, the recombinase comprises the sequence of Bxb1 recombinase, and the corresponding recombinase recognition sequences are Bxb1 attB and Bxb1 attP. 
     In some embodiments, the recombinase comprises the sequence of phiC31 (ϕC31) recombinase and the corresponding recombinase recognition sequences comprise phiC31 attB and phiC31 attP. 
     A recombinase can recognize multiple pairs of RRS. In some embodiments, the recombinase comprises the sequence of Cre and the corresponding recombinase recognition sequences comprise loxP. In some embodiments, the recombinase comprises the sequence of Cre and the corresponding recombinase recognition sequences comprise lox2272. In some embodiments, the recombinase comprises the sequence of Cre and the corresponding recombinase recognition sequences comprise loxN. 
     In some embodiments, the recombinase comprises the sequence of Dre and the corresponding recombinase recognition sequences comprise rox. 
     In some embodiments, the recombinase comprises the sequence of VCre and the corresponding recombinase recognition sequences comprise VloxP. 
     In some embodiments, the recombinase comprises the sequence of VCre and the corresponding recombinase recognition sequences comprise VloxP. 
     In some embodiments, the recombinase comprises the sequence of Flp and the corresponding recombinase recognition sequences comprise FRT. 
     In some embodiments, the recombinase comprises the sequence of SCre and the corresponding recombinase recognition sequences comprise SloxM1. 
     In some embodiments, the recombinase comprises the sequence of Vika and the corresponding recombinase recognition sequences comprise vox. 
     In some embodiments, the recombinase comprises the sequence of B3 and the corresponding recombinase recognition sequences comprise B3RT. 
     In some embodiments, the recombinase comprises the sequence of KD and the corresponding recombinase recognition sequences comprise KDRT. 
     The sequences for some recombinases are shown below: 
     
       
         
           
               
            
               
                 hPGK (SEQ ID NO: 1): 
               
               
                 ggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggac 
               
               
                   
               
               
                 gcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctggg 
               
               
                   
               
               
                 tctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgc 
               
               
                   
               
               
                 tacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcggg 
               
               
                   
               
               
                 aaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgca 
               
               
                   
               
               
                 cgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagc 
               
               
                   
               
               
                 gcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcg 
               
               
                   
               
               
                 ccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcg 
               
               
                   
               
               
                 gtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcc 
               
               
                   
               
               
                 tccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacc 
               
               
                   
               
               
                 tctctccccag 
               
               
                   
               
               
                 EF1alpha (SEQ ID NO: 2): 
               
               
                 cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtc 
               
               
                   
               
               
                 cccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaag 
               
               
                   
               
               
                 gtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgccttt 
               
               
                   
               
               
                 ttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaac 
               
               
                   
               
               
                 gttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtg 
               
               
                   
               
               
                 tggttcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt 
               
               
                   
               
               
                 gaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgg 
               
               
                   
               
               
                 gttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttc 
               
               
                   
               
               
                 gcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgc 
               
               
                   
               
               
                 gaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctcta 
               
               
                   
               
               
                 gccatttaaaatttttgatgacctgctgcgacgctttttttctggcaaga 
               
               
                   
               
               
                 tagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttg 
               
               
                   
               
               
                 gggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcg 
               
               
                   
               
               
                 aggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctca 
               
               
                   
               
               
                 agctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccc 
               
               
                   
               
               
                 cgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaa 
               
               
                   
               
               
                 agatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcg 
               
               
                   
               
               
                 gcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcct 
               
               
                   
               
               
                 ttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccg 
               
               
                   
               
               
                 tccaggcacctcgattagttctcgagcttttggagtacgtcgtctttagg 
               
               
                   
               
               
                 ttggggggaggggttttatgcgatggagtttccccacactgagtgggtgg 
               
               
                   
               
               
                 agactgaagttaggccagcttggcacttgatgtaattctccttggaattt 
               
               
                   
               
               
                 gccctttttgagtttggatcttggttcattctcaagcctcagacagtggt 
               
               
                   
               
               
                 tcaaagtttttttcttccatttcaggtgtcgtga 
               
               
                   
               
               
                 SFFV (SEQ ID NO: 3): 
               
               
                 ccgataaaataaaagattttatttagtctccagaaaaaggggggaatgaa 
               
               
                   
               
               
                 agaccccacctgtaggtttggcaagctagctgcagtaacgccattttgca 
               
               
                   
               
               
                 aggcatggaaaaataccaaaccaagaatagagaagttcagatcaagggcg 
               
               
                   
               
               
                 ggtacatgaaaatagctaacgttgggccaaacaggatatctgcggtgagc 
               
               
                   
               
               
                 agtttcggccccggcccggggccaagaacagatggtcaccgcagtttcgg 
               
               
                   
               
               
                 ccccggcccgaggccaagaacagatggtccccagatatggcccaaccctc 
               
               
                   
               
               
                 agcagtttcttaagacccatcagatgtttccaggctcccccaaggacctg 
               
               
                   
               
               
                 aaatgaccctgcgccttatttgaattaaccaatcagcctgcttctcgctt 
               
               
                   
               
               
                 ctgttcgcgcgcttctgcttcccgagctctataaaagagctcacaacccc 
               
               
                   
               
               
                 tcactcggcgcgccagtcctccgacagactgagtcgcccggg 
               
               
                   
               
               
                 CAG (SEQ ID NO: 4): 
               
               
                 ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA 
               
               
                   
               
               
                 TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGAC 
               
               
                   
               
               
                 CGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATA 
               
               
                   
               
               
                 GTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACG 
               
               
                   
               
               
                 GTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGC 
               
               
                   
               
               
                 CCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG 
               
               
                   
               
               
                 TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT 
               
               
                   
               
               
                 CATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC 
               
               
                   
               
               
                 CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTA 
               
               
                   
               
               
                 ATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGC 
               
               
                   
               
               
                 GGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGC 
               
               
                   
               
               
                 GGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC 
               
               
                   
               
               
                 GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTC 
               
               
                   
               
               
                 GCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCC 
               
               
                   
               
               
                 GCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACG 
               
               
                   
               
               
                 GCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTT 
               
               
                   
               
               
                 TCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTT 
               
               
                   
               
               
                 GTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGG 
               
               
                   
               
               
                 AGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGC 
               
               
                   
               
               
                 GGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGG 
               
               
                   
               
               
                 GGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTG 
               
               
                   
               
               
                 CGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGG 
               
               
                   
               
               
                 GCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCG 
               
               
                   
               
               
                 GCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCC 
               
               
                   
               
               
                 GGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTC 
               
               
                   
               
               
                 GGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCG 
               
               
                   
               
               
                 GCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGC 
               
               
                   
               
               
                 GAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCT 
               
               
                   
               
               
                 GGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGC 
               
               
                   
               
               
                 GCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCG 
               
               
                   
               
               
                 CCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTG 
               
               
                   
               
               
                 CCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCG 
               
               
                   
               
               
                 GCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTA 
               
               
                   
               
               
                 CAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAA 
               
               
                   
               
               
                 A 
               
               
                   
               
               
                 NLS-iCre (SEQ ID NO: 5): 
               
               
                 atggtgcccaagaagaagaggaaagtctccaacctgctgactgtgcacca 
               
               
                   
               
               
                 aaacctgcctgccctccctgtggatgccacctctgatgaagtcaggaaga 
               
               
                   
               
               
                 acctgatggacatgttcagggacaggcaggccttctctgaacacacctgg 
               
               
                   
               
               
                 aagatgctcctgtctgtgtgcagatcctgggctgcctggtgcaagctgaa 
               
               
                   
               
               
                 caacaggaaatggttccctgctgaacctgaggatgtgagggactacctcc 
               
               
                   
               
               
                 tgtacctgcaagccagaggcctggctgtgaagaccatccaacagcacctg 
               
               
                   
               
               
                 ggccagctcaacatgctgcacaggagatctggcctgcctcgcccttctga 
               
               
                   
               
               
                 ctccaatgctgtgtccctggtgatgaggagaatcagaaaggagaatgtgg 
               
               
                   
               
               
                 atgctggggagagagccaagcaggccctggcctttgaacgcactgacttt 
               
               
                   
               
               
                 gaccaagtcagatccctgatggagaactctgacagatgccaggacatcag 
               
               
                   
               
               
                 gaacctggccttcctgggcattgcctacaacaccctgctgcgcattgccg 
               
               
                   
               
               
                 aaattgccagaatcagagtgaaggacatctcccgcaccgatggtgggaga 
               
               
                   
               
               
                 atgctgatccacattggcaggaccaagaccctggtgtccacagctggtgt 
               
               
                   
               
               
                 ggagaaggccctgtccctgggggttaccaagctggtggagagatggatct 
               
               
                   
               
               
                 ctgtgtctggtgtggctgatgaccccaacaactacctgttctgccgggtc 
               
               
                   
               
               
                 agaaagaatggtgtggctgccccttctgccacctcccaactgtccacccg 
               
               
                   
               
               
                 ggccctggaagggatctttgaggccacccaccgcctgatctatggtgcca 
               
               
                   
               
               
                 aggatgactctgggcagagatacctggcctggtctggccactctgccaga 
               
               
                   
               
               
                 gtgggtgctgccagggacatggccagggctggtgtgtccatccctgaaat 
               
               
                   
               
               
                 catgcaggctggtggctggaccaatgtgaacattgtgatgaactacatca 
               
               
                   
               
               
                 gaaacctggactctgagactggggccatggtgaggctgctcgaggatggg 
               
               
                   
               
               
                 gactga 
               
               
                   
               
               
                 NLS-FlpO (SEQ ID NO: 6): 
               
               
                 atggctcctaagaagaagaggaaggtgatgagccagttcgacatcctgtg 
               
               
                   
               
               
                 caagaccccccccaaggtgctggtgcggcagttcgtggagagattcgaga 
               
               
                   
               
               
                 ggcccagcggcgagaagatcgccagctgtgccgccgagctgacctacctg 
               
               
                   
               
               
                 tgctggatgatcacccacaacggcaccgccatcaagagggccaccttcat 
               
               
                   
               
               
                 gagctacaacaccatcatcagcaacagcctgagcttcgacatcgtgaaca 
               
               
                   
               
               
                 agagcctgcagttcaagtacaagacccagaaggccaccatcctggaggcc 
               
               
                   
               
               
                 agcctgaagaagctgatccccgcctgggagttcaccatcatcccttacaa 
               
               
                   
               
               
                 cggccagaagcaccagagcgacatcaccgacatcgtgtccagcctgcagc 
               
               
                   
               
               
                 tgcagttcgagagcagcgaggaggccgacaagggcaacagccacagcaag 
               
               
                   
               
               
                 aagatgctgaaggccctgctgtccgagggcgagagcatctgggagatcac 
               
               
                   
               
               
                 cgagaagatcctgaacagcttcgagtacaccagcaggttcaccaagacca 
               
               
                   
               
               
                 agaccctgtaccagttcctgttcctggccacattcatcaactgcggcagg 
               
               
                   
               
               
                 ttcagcgacatcaagaacgtggaccccaagagcttcaagctggtgcagaa 
               
               
                   
               
               
                 caagtacctgggcgtgatcattcagtgcctggtgaccgagaccaagacaa 
               
               
                   
               
               
                 gcgtgtccaggcacatctactttttcagcgccagaggcaggatcgacccc 
               
               
                   
               
               
                 ctggtgtacctggacgagttcctgaggaacagcgagcccgtgctgaagag 
               
               
                   
               
               
                 agtgaacaggaccggcaacagcagcagcaacaagcaggagtaccagctgc 
               
               
                   
               
               
                 tgaaggacaacctggtgcgcagctacaacaaggccctgaagaagaacgcc 
               
               
                   
               
               
                 ccctaccccatcttcgctatcaagaacggccctaagagccacatcggcag 
               
               
                   
               
               
                 gcacctgatgaccagctttctgagcatgaagggcctgaccgagctgacaa 
               
               
                   
               
               
                 acgtggtgggcaactggagcgacaagagggcctccgccgtggccaggacc 
               
               
                   
               
               
                 acctacacccaccagatcaccgccatccccgaccactacttcgccctggt 
               
               
                   
               
               
                 gtccaggtactacgcctacgaccccatcagcaaggagatgatcgccctga 
               
               
                   
               
               
                 aggacgagaccaaccccatcgaggagtggcagcacatcgagcagctgaag 
               
               
                   
               
               
                 ggcagcgccgagggcagcatcagataccccgcctggaacggcatcatcag 
               
               
                   
               
               
                 ccaggaggtgctggactacctgagcagctacatcaacaggcggatctga 
               
               
                   
               
               
                 NLS-DreO (SEQ ID NO: 7): 
               
               
                 ATGCCTAAGAAGAAGAGGAAGGTTTCTGAGCTGATTATTAGTGGTTCATC 
               
               
                   
               
               
                 TGGTGGATTCCTGCGAAACATCGGCAAAGAGTATCAGGAGGCCGCTGAAA 
               
               
                   
               
               
                 ACTTCATGAGGTTTATGAATGACCAGGGGGCGTACGCTCCTAACACTTTG 
               
               
                   
               
               
                 AGGGATTTGAGGTTGGTCTTTCATAGCTGGGCCAGATGGTGCCATGCTCG 
               
               
                   
               
               
                 GCAGCTTGCATGGTTTCCAATTAGTCCTGAAATGGCACGCGAATACTTTC 
               
               
                   
               
               
                 TTCAGTTGCACGATGCAGACCTGGCCTCCACTACCATCGACAAGCACTAT 
               
               
                   
               
               
                 GCTATGCTTAATATGCTTCTGTCCCACTGCGGACTGCCACCCTTGTCCGA 
               
               
                   
               
               
                 CGACAAGTCAGTGAGTCTTGCCATGAGAAGAATTAGAAGAGAAGCCGCAA 
               
               
                   
               
               
                 CCGAAAAGGGTGAGAGGACAGGACAGGCAATCCCCCTGCGCTGGGACGAC 
               
               
                   
               
               
                 CTGAAGCTGCTGGATGTGCTGCTCAGCAGGAGCGAGCGGCTGGTCGACCT 
               
               
                   
               
               
                 GCGCAACAGGGCTTTCCTGTTCGTAGCCTATAACACCCTCATGAGAATGT 
               
               
                   
               
               
                 CTGAAATATCACGCATCAGGGTTGGGGACTTGGATCAGACAGGAGACACA 
               
               
                   
               
               
                 GTGACCCTGCACATCAGTCACACTAAGACAATCACCACAGCTGCGGGCCT 
               
               
                   
               
               
                 TGACAAAGTGCTCTCCCGGCGAACCACAGCAGTGCTCAATGACTGGCTGG 
               
               
                   
               
               
                 ACGTCAGTGGGCTTAGAGAACATCCAGACGCTGTGCTCTTCCCACCTATA 
               
               
                   
               
               
                 CACCGGTCAAACAAAGCCCGCATTACTACCACGCCCCTGACCGCCCCTGC 
               
               
                   
               
               
                 CATGGAGAAGATTTTCAGTGATGCCTGGGTGCTGCTGAACAAACGGGACG 
               
               
                   
               
               
                 CCACCCCCAATAAAGGGAGGTATAGGACCTGGACCGGCCATTCCGCCAGG 
               
               
                   
               
               
                 GTGGGTGCCGCAATAGACATGGCCGAGAAACAGGTGTCTATGGTCGAGAT 
               
               
                   
               
               
                 TATGCAGGAAGGGACATGGAAGAAGCCTGAAACACTGATGCGGTATCTCA 
               
               
                   
               
               
                 GAAGGGGCGGAGTGTCCGTGGGAGCCAATTCTCGACTGATGGATAGCTAA 
               
               
                   
               
               
                 NLS-SCre (SEQ ID NO: 8): 
               
               
                 ATGCCCAAGAAAAAGCGGAAGGTGTCCCTGCTGACCACCAACAACCACAG 
               
               
                   
               
               
                 CGTGGCCCTGAGCTACGGCGAGCCTCCTAGCACCCTGAACGACAGCCTGA 
               
               
                   
               
               
                 AGGACAGCTACCAGCGGAGCACCGATGAGCTGCAGGCCCTGCTGTCTAAG 
               
               
                   
               
               
                 CCTCTGGCCCAGCTGACCGACGCCGACAAGCTGCGGATCAGAGAGATCAC 
               
               
                   
               
               
                 CCAGGCCAAGCTGAAGCACTTCCTGGACAACGGCCACCGGACCAGAAGGG 
               
               
                   
               
               
                 CCAACACTTGGAGAGCCCTGATGAGCAGATGGGCCAAGTTCGAGAGCTGG 
               
               
                   
               
               
                 TGCCTGACCAACAATCTGACCCCCCTGCCTGCCACCCCTGAGGTGGTGGC 
               
               
                   
               
               
                 CACATTCATCGAGTACTACCAGGCCAGCAGCTACACCACCCTGAGCCAGT 
               
               
                   
               
               
                 ATGCCTGGGCCATCAACAGCTTTCACGTGGAATGCGGCCTGCTGAGCCCC 
               
               
                   
               
               
                 GTGTCTAGCAAGACCGTGCAGGACAAGCAGAACGAGATCAGAATCGTGAA 
               
               
                   
               
               
                 GCTGGAATCTGGCGGCCTGGCCCAGGAACAGGCCACCCCTTTTAGACTGC 
               
               
                   
               
               
                 ACCATCTGCAGATGCTGATCGAGAGCTATGGCGAGAGCGAGCGGCTGCTG 
               
               
                   
               
               
                 GACAAGAGAAACCTGGCTCTGCTGAATATCGCCTACGAGAGCCTGCTGCG 
               
               
                   
               
               
                 CGAGTCCGAGCTGCTGAGAATCAAAGTGGGCCACCTGAAGTCCACCTTCG 
               
               
                   
               
               
                 AGGGCGACTACGTGCTGAGCGTGCCCTACACCAAGACCAACGACAGCGGC 
               
               
                   
               
               
                 GAAGAGGAAGTCGTGAACATCACCCCCCTGGGCTTCAAGCTGATCCAGCG 
               
               
                   
               
               
                 GTACATCCAGGGCGCTGGCCTGACAAAAGAGGACTACCTGTTCCAGCCCA 
               
               
                   
               
               
                 TCGGCCGGTCCAACAAGGTGTCCGTGCAGGCCAAACCCATGAGCACCCGG 
               
               
                   
               
               
                 ACCGTGGACAGAGTGTTCCTGTGGGCCTTTGAGAGCCTGGGCATCGACAG 
               
               
                   
               
               
                 ACACAGCGCTTGGAGCGGCCACAGCGCCAGAATTGGAGCCGCTCAGGATC 
               
               
                   
               
               
                 TGCTGGCCGCTGGCTATTCTATCGCCCAGATCCAGGAAAACGGCCGCTGG 
               
               
                   
               
               
                 AAGTCCCCCATGATGGTGCTGAGATACGGCAAGGACATCAAGGCCAAAGA 
               
               
                   
               
               
                 AAGCGCCATGGCCAAGATGCTGGCCGAGCGGAGATGA 
               
               
                   
               
               
                 NLS-VCre (SEQ ID NO: 9): 
               
               
                 ATGCCCAAGAAAAAGCGGAAAGTGATCGAGAACCAGCTGAGCCTGCTGGG 
               
               
                   
               
               
                 CGACTTTTCTGGCGTGCGGCCCGACGATGTGAAAACCGCCATTCAGGCCG 
               
               
                   
               
               
                 CCCAGAAAAAGGGCATCAACGTGGCCGAGAACGAGCAGTTCAAGGCCGCC 
               
               
                   
               
               
                 TTCGAGCATCTGCTGAACGAGTTCAAGAAGCGGGAAGAGAGATACAGCCC 
               
               
                   
               
               
                 CAACACCCTGCGGCGGCTGGAAAGCGCCTGGACCTGCTTCGTGGATTGGT 
               
               
                   
               
               
                 GCCTGGCCAACCACAGACACAGCCTGCCTGCCACCCCCGATACCGTGGAA 
               
               
                   
               
               
                 GCCTTCTTCATCGAGCGGGCCGAGGAACTGCACCGGAACACCCTGAGCGT 
               
               
                   
               
               
                 GTACAGATGGGCCATCAGCCGGGTGCACAGAGTGGCCGGATGCCCTGATC 
               
               
                   
               
               
                 CCTGCCTGGACATCTACGTGGAAGATCGGCTGAAGGCCATTGCCCGGAAG 
               
               
                   
               
               
                 AAAGTGCGGGAAGGCGAGGCCGTGAAGCAGGCCAGCCCTTTCAACGAGCA 
               
               
                   
               
               
                 GCATCTGCTGAAGCTGACCAGCCTGTGGTACAGAAGCGACAAGCTGCTGC 
               
               
                   
               
               
                 TGCGGCGGAACCTGGCTCTGCTGGCTGTGGCCTACGAGAGCATGCTGAGA 
               
               
                   
               
               
                 GCCAGCGAGCTGGCCAACATCCGGGTGTCCGATATGGAACTGGCCGGCGA 
               
               
                   
               
               
                 CGGAACCGCCATCCTGACCATCCCTATCACCAAGACCAACCACTCCGGCG 
               
               
                   
               
               
                 AGCCCGATACCTGCATCCTGTCCCAGGATGTGGTGTCCCTGCTGATGGAC 
               
               
                   
               
               
                 TACACCGAGGCCGGCAAGCTGGATATGAGCAGCGACGGCTTCCTGTTCGT 
               
               
                   
               
               
                 GGGCGTGTCCAAGCACAACACCTGTATCAAGCCCAAGAAGGACAAGCAGA 
               
               
                   
               
               
                 CCGGCGAGGTGCTGCACAAGCCCATCACCACCAAGACAGTGGAAGGCGTG 
               
               
                   
               
               
                 TTCTACAGCGCCTGGGAGACACTGGACCTGGGCAGACAGGGCGTGAAGCC 
               
               
                   
               
               
                 TTTCACAGCCCACAGCGCCAGAGTGGGAGCCGCTCAGGACCTGCTGAAGA 
               
               
                   
               
               
                 AGGGCTACAATACCCTGCAGATCCAGCAGTCCGGCCGGTGGTCTAGCGGA 
               
               
                   
               
               
                 GCCATGGTGGCCAGATACGGCAGAGCCATCCTGGCTAGGGATGGCGCTAT 
               
               
                   
               
               
                 GGCCCACAGCAGAGTGAAAACCAGATCCGCCCCCATGCAGTGGGGCAAGG 
               
               
                   
               
               
                 ACGAGAAGGACTGA 
               
               
                   
               
               
                 NLS-VikaO (SEQ ID NO: 10): 
               
               
                 ATGCCCAAGAAAAAGCGGAAAGTGACCGACCTGACCCCATTCCCCCCCCT 
               
               
                   
               
               
                 GGAACACCTGGAACCCGACGAGTTTGCCGACCTCGTGCGGAAGGCCATCA 
               
               
                   
               
               
                 AGAGGGATCCTCAGGCTGGCGCCCACCCTGCCATCCAGTCTGCCATCAGC 
               
               
                   
               
               
                 CACTTCCAGGACGAGTTCGTGCGGAGACAGGGCGAATGGCAGCCTGCCAC 
               
               
                   
               
               
                 ACTGCAGAGACTGAGAAACGCCTGGAATGTGTTTGTGCGGTGGTGCACCC 
               
               
                   
               
               
                 ACCAGGGCATTCCAGCTCTGCCTGCCAGACACCAGGACGTGGAAAGATAC 
               
               
                   
               
               
                 CTGATCGAGCGGCGGAACGAGCTGCACCGGAACACCCTGAAAGTGCACCT 
               
               
                   
               
               
                 GTGGGCCATCGGCAAGACCCACGTGATCAGCGGCCTGCCCAATCCCTGCG 
               
               
                   
               
               
                 CCCACAGATACGTGAAAGCCCAGATGGCCCAGATCACACACCAGAAAGTG 
               
               
                   
               
               
                 CGCGAGAGAGAGCGGATCGAACAGGCCCCTGCCTTCAGAGAGTCCGACCT 
               
               
                   
               
               
                 GGACAGACTGACCGAGCTGTGGAGCGCCACCAGAAGCGTGACCCAGCAGC 
               
               
                   
               
               
                 GGGACCTGATGATCGTGTCCCTGGCCTACGAGACACTGCTGCGGAAGAAC 
               
               
                   
               
               
                 AATCTGGAACAGATGAAAGTGGGCGACATCGAGTTCTGCCAGGACGGCTC 
               
               
                   
               
               
                 TGCCCTGATCACCATCCCCTTCAGCAAGACCAACCACAGCGGCAGGGATG 
               
               
                   
               
               
                 ACGTGCGGTGGATCTCTCCCCAGGTGGCCAATCAGGTGCACGCCTACCTG 
               
               
                   
               
               
                 CAGCTGCCCAACATCGACGCCGACCCCCAGTGCTTCCTGCTGCAGAGAGT 
               
               
                   
               
               
                 GAAGAGAAGCGGCAAGGCCCTGAACCCCGAGAGCCACAATACCCTGAACG 
               
               
                   
               
               
                 GCCACCACCCCGTGTCCGAGAAGCTGATCTCCCGGGTGTTCGAGCGGGCT 
               
               
                   
               
               
                 TGGAGAGCCCTGAATCACGAGACAGGCCCCAGATACACCGGCCACAGCGC 
               
               
                   
               
               
                 TAGAGTGGGAGCCGCTCAGGATCTGCTGCAGGAAGGCTACAGCACCCTGC 
               
               
                   
               
               
                 AAGTGATGCAGGCAGGCGGCTGGTCCAGCGAGAAGATGGTGCTGAGATAC 
               
               
                   
               
               
                 GGCCGGCATCTGCACGCCCACACATCTGCCATGGCTCAGAAACGGCGGCA 
               
               
                   
               
               
                 GCGGTGA 
               
               
                   
               
               
                 NLS-B3 (SEQ ID NO: 11): 
               
               
                 ATGCCCAAGAAAAAGCGGAAGGTGTCCAGCTACATGGACCTGGTGGACGA 
               
               
                   
               
               
                 CGAGCCCGCCACCCTGTACCACAAGTTCGTGGAATGCCTGAAGGCCGGCG 
               
               
                   
               
               
                 AGAACTTCTGCGGCGATAAGCTGAGCGGCATCATCACCATGGCCATTCTG 
               
               
                   
               
               
                 AAGGCCATCAAGGCCCTGACCGAAGTGAAGAAAACCACCTTCAACAAGTA 
               
               
                   
               
               
                 CAAGACCACCATCAAGCAGGGCCTGCAGTACGACGTGGGCAGCAGCACCA 
               
               
                   
               
               
                 TCAGCTTCGTGTACCACCTGAAGGACTGCGACGAGCTGAGCAGAGGCCTG 
               
               
                   
               
               
                 AGCGACGCCTTCGAGCCCTACAAGTTCAAGATCAAGAGCAACAAAGAGGC 
               
               
                   
               
               
                 CACCAGCTTCAAGACCCTGTTCAGGGGCCCTAGCTTCGGCAGCCAGAAGA 
               
               
                   
               
               
                 ACTGGCGGAAGAAAGAGGTGGACCGCGAGGTGGACAACCTGTTCCACAGC 
               
               
                   
               
               
                 ACCGAGACAGACGAGAGCATCTTCAAGTTCATCCTGAACACCCTGGACAG 
               
               
                   
               
               
                 CATCGAAACCCAGACCAACACCGACCGGCAGAAAACCGTGCTGACCTTTA 
               
               
                   
               
               
                 TCCTGCTGATGACCTTCTTCAACTGCTGCCGGAACAACGACCTGATGAAC 
               
               
                   
               
               
                 GTGGACCCCAGCACCTTCAAGATCGTGAAGAACAAGTTTGTGGGCTACCT 
               
               
                   
               
               
                 GCTGCAGGCTGAAGTGAAGCAGACCAAGACCAGAAAGAGCCGGAATATCT 
               
               
                   
               
               
                 TCTTCTTCCCCATCCGGGAAAACCGCTTCGACCTGTTCCTGGCCCTGCAC 
               
               
                   
               
               
                 GACTTCTTCAGAACCTGCCAGCCCACCCCCAAGAGCAGACTGAGCGATCA 
               
               
                   
               
               
                 GGTGTCCGAGCAGAAGTGGCAGCTGTTCCGGGACAGCATGGTCATCGACT 
               
               
                   
               
               
                 ACAACCGGTTCTTTCGGAAGTTCCCCGCCAGCCCCATCTTCGCCATTAAG 
               
               
                   
               
               
                 CACGGCCCCAAGTCCCACCTGGGCCGGCATCTGATGAACAGCTTTCTGCA 
               
               
                   
               
               
                 CAAGAACGAGCTGGACAGCTGGGCCAACAGCCTGGGCAATTGGAGCAGCT 
               
               
                   
               
               
                 CCCAGAACCAGAGAGAGAGCGGCGCCAGACTGGGCTACACACACGGCGGA 
               
               
                   
               
               
                 AGAGATCTGCCCCAGCCCCTGTTTGGCTTCCTGGCCGGATACTGCGTGCG 
               
               
                   
               
               
                 GAACGAAGAGGGCCACATCGTGGGCCTGGGCCTGGAAAAGGACATCAACG 
               
               
                   
               
               
                 ATCTGTTCGACGGCATCATGGACCCCCTGAACGAGAAAGAGGACACCGAG 
               
               
                   
               
               
                 ATCTGCGAGAGCTACGGCGAGTGGGCCAAGATTGTGTCCAAGGACGTGCT 
               
               
                   
               
               
                 GATCTTCCTGAAGAGATACCACAGCAAGAACGCCTGTCGGAGATACCAGA 
               
               
                   
               
               
                 ACAGCACCCTGTATGCCCGGACCTTCCTGAAAACCGAGAGCGTGACCCTG 
               
               
                   
               
               
                 AGCGGCTCCAAGGGCAGCGAGGAACCTTCTAGCCCTGTGCGGATCCCCAT 
               
               
                   
               
               
                 CCTGAGCATGGGAAAGGCCAGCCCCTCCGAGGGAAGAAAGCTGAGAGCCA 
               
               
                   
               
               
                 GCGAGCACGCCAACGACGACAACGAGATCGAGAAGATCGACAGCGACAGC 
               
               
                   
               
               
                 AGCCAGAGCGAAGAGATCCCTATCGAGATGAGCGACTCCGAGGACGAGAC 
               
               
                   
               
               
                 AACCGCCAGCAACATCAGCGGCATCTACCTGGACATGAGCAAGGCCAACT 
               
               
                   
               
               
                 CCAACGTGGTGTACAGCCCCCCTAGCCAGACAGGCAGAGCTGCTGGCGCC 
               
               
                   
               
               
                 GGAAGAAAAAGAGGCGTGGGAGGCAGACGGACCGTGGAAAGCAAGCGGAG 
               
               
                   
               
               
                 AAGAGTGCTGGCCCCCATCAACCGGTGA 
               
               
                   
               
               
                 NLS-KD (SEQ ID NO: 12): 
               
               
                 ATGCCCAAGAAAAAGCGGAAGGTGTCCACCTTCGCCGAGGCCGCCCATCT 
               
               
                   
               
               
                 GACACCTCACCAGTGCGCCAACGAGATCAATGAGATCCTGGAAAGCGACA 
               
               
                   
               
               
                 CCTTCAACATCAACGCCAAAGAGATCCGGAACAAGCTGGCCTCCCTGTTC 
               
               
                   
               
               
                 AGCATCCTGACCATGCAGAGCCTGAGCATCCGCAGAGAGATGAAGATCAA 
               
               
                   
               
               
                 CACCTACCGGTCCTACAAGAGCGCCATCGGCAAGAGCCTGTCCTTCGACA 
               
               
                   
               
               
                 AGGACGACAAGATCATCAAGTTCACCGTGCGGCTGAGAAAGACCGAGAGC 
               
               
                   
               
               
                 CTGCAGAAGGACATCGAGAGCGCCCTGCCCAGCTACAAGGTGGTGGTGTC 
               
               
                   
               
               
                 CCCATTCAAGAACCAGGAAGTGTCCCTGTTCGACCGCTACGAGGAAACCC 
               
               
                   
               
               
                 ACAAATACGACGCCAGCATGGTGGGACTGCAGTTCACCAACATCCTGAGC 
               
               
                   
               
               
                 AAAGAGAAGGATATCTGGAAGATCGTGTCCCGGATCGCCTGCTTCTTCGA 
               
               
                   
               
               
                 CCAGAGCTGCGTGACCACCACCAAGCGGGCCGAGTACAGACTGCTGCTGC 
               
               
                   
               
               
                 TGGGCGCTGTGGGCAACTGCTGCAGATACAGCGACCTGAAGAACCTGGAC 
               
               
                   
               
               
                 CCCCGGACCTTCGAGATCTACAACAACAGCTTCCTGGGCCCCATCGTGCG 
               
               
                   
               
               
                 GGCCACCGTGACAGAGACAAAGAGCCGGACCGAGAGATACGTGAACTTCT 
               
               
                   
               
               
                 ACCCCGTGAACGGCGACTGCGACCTGCTGATCTCCCTGTACGACTACCTG 
               
               
                   
               
               
                 AGAGTGTGCAGCCCCATCGAGAAAACCGTGTCCAGCAACCGGCCCACCAA 
               
               
                   
               
               
                 CCAGACCCACCAGTTTCTGCCTGAGAGCCTGGCCAGAACCTTCAGCCGGT 
               
               
                   
               
               
                 TCCTGACCCAGCACGTGGACGAGCCCGTGTTCAAGATCTGGAACGGCCCC 
               
               
                   
               
               
                 AAGAGCCACTTCGGCAGACACCTGATGGCCACCTTTCTGAGCAGAAGCGA 
               
               
                   
               
               
                 GAAGGGCAAATACGTGTCCTCCCTGGGCAATTGGGCTGGCGACCGGGAAA 
               
               
                   
               
               
                 TCCAGTCTGCCGTGGCCAGAAGCCACTACAGCCACGGCTCTGTGACCGTG 
               
               
                   
               
               
                 GACGACCGGGTGTTCGCCTTCATCAGCGGCTTCTACAAAGAGGCCCCCCT 
               
               
                   
               
               
                 GGGCAGCGAGATCTATGTGCTGAAGGACCCCAGCAACAAGCCCCTGAGCA 
               
               
                   
               
               
                 GAGAGGAACTGCTGGAAGAGGAAGGCAACAGCCTGGGCTCCCCACCTCTG 
               
               
                   
               
               
                 AGCCCTCCAAGCTCTCCTAGACTGGTGGCCCAGAGCTTCAGCGCCCACCC 
               
               
                   
               
               
                 AAGCCTGCAGCTGTTCGAGCAGTGGCACGGCATCATCAGCGACGAGGTGC 
               
               
                   
               
               
                 TGCAGTTTATCGCCGAGTACCGGCGGAAGCACGAGCTGAGAAGCCAGAGA 
               
               
                   
               
               
                 ACCGTGGTGGCCTGA 
               
               
                   
               
               
                 NLS-B2 (SEQ ID NO: 13): 
               
               
                 ATGCCCAAGAAAAAGCGGAAGGTGTCCGAGTTCAGCGAGCTCGTGCGGAT 
               
               
                   
               
               
                 CCTGCCCCTGGATCAGGTGGCCGAGATCAAGAGAATCCTGAGCAGAGGCG 
               
               
                   
               
               
                 ACCCCATCCCCCTGCAGAGACTGGCCTCTCTGCTGACCATGGTCATCCTG 
               
               
                   
               
               
                 ACCGTGAACATGAGCAAGAAGAGAAAGAGCAGCCCCATCAAGCTGAGCAC 
               
               
                   
               
               
                 CTTCACCAAGTACCGGCGGAACGTGGCCAAGAGCCTGTACTACGACATGA 
               
               
                   
               
               
                 GCAGCAAGACCGTGTTCTTCGAGTACCACCTGAAGAACACCCAGGACCTG 
               
               
                   
               
               
                 CAGGAAGGCCTGGAACAGGCCATTGCCCCCTACAACTTCGTCGTGAAAGT 
               
               
                   
               
               
                 GCACAAGAAGCCCATCGACTGGCAGAAACAGCTGAGCAGCGTGCACGAGC 
               
               
                   
               
               
                 GGAAGGCCGGCCACAGATCCATCCTGTCCAACAACGTGGGCGCCGAGATC 
               
               
                   
               
               
                 TCCAAGCTGGCCGAGACAAAGGACAGCACCTGGTCCTTCATCGAGCGGAC 
               
               
                   
               
               
                 CATGGACCTGATCGAGGCCAGAACCAGACAGCCCACCACCAGAGTGGCCT 
               
               
                   
               
               
                 ACCGGTTCCTGCTGCAGCTGACCTTCATGAACTGCTGCCGGGCCAACGAT 
               
               
                   
               
               
                 CTGAAGAACGCCGACCCCAGCACCTTCCAGATCATTGCCGATCCCCACCT 
               
               
                   
               
               
                 GGGCCGGATCCTGAGAGCCTTCGTGCCCGAGACTAAGACCTCTATCGAGC 
               
               
                   
               
               
                 GGTTTATCTACTTCTTCCCATGCAAGGGCCGCTGCGACCCTCTGCTGGCC 
               
               
                   
               
               
                 CTGGATTCTTACCTGCTGTGGGTGGGACCCGTGCCCAAGACCCAGACCAC 
               
               
                   
               
               
                 CGATGAGGAAACCCAGTACGACTACCAGCTGCTGCAGGACACCCTGCTGA 
               
               
                   
               
               
                 TCTCTTACGACCGGTTTATCGCCAAAGAGAGCAAAGAGAACATCTTCAAG 
               
               
                   
               
               
                 ATCCCCAACGGCCCCAAGGCCCATCTGGGCAGACATCTGATGGCCAGCTA 
               
               
                   
               
               
                 CCTGGGCAACAACAGCCTGAAGTCCGAGGCCACCCTGTACGGCAATTGGA 
               
               
                   
               
               
                 GCGTGGAAAGACAGGAAGGCGTGTCCAAAATGGCCGACAGCCGGTACATG 
               
               
                   
               
               
                 CACACCGTGAAGAAGTCCCCCCCCTCCTACCTGTTCGCCTTTCTGAGCGG 
               
               
                   
               
               
                 CTACTACAAGAAGTCCAACCAGGGCGAGTACGTGCTGGCCGAAACCCTGT 
               
               
                   
               
               
                 ACAACCCCCTGGACTACGATAAGACCCTGCCCATCACCACCAACGAGAAG 
               
               
                   
               
               
                 CTGATCTGCAGACGCTACGGCAAGAACGCCAAAGTGATCCCCAAGGATGC 
               
               
                   
               
               
                 CCTGCTGTACCTGTACACCTACGCCCAGCAGAAGCGGAAGCAGCTGGCTG 
               
               
                   
               
               
                 ACCCCAACGAGCAGAACCGGCTGTTCAGCAGCGAGAGCCCTGCCCACCCA 
               
               
                   
               
               
                 TTTCTGACCCCTCAGAGCACAGGCAGCAGCACCCCTCTGACATGGACCGC 
               
               
                   
               
               
                 CCCTAAGACACTGAGCACCGGCCTGATGACCCCTGGCGAGGAATGA 
               
               
                   
               
               
                 NLS-R (SEQ ID NO: 14): 
               
               
                 ATGCCCAAGAAAAAGCGGAAGGTGCAGCTGACCAAGGACACCGAGATCAG 
               
               
                   
               
               
                 CACCATCAACCGGCAGATGAGCGACTTCAGCGAGCTGAGCCAGATCCTGC 
               
               
                   
               
               
                 CCCTGCACCAGATCTCCAAGATCAAGGACATCCTGGAAAACGAGAACCCC 
               
               
                   
               
               
                 CTGCCCAAAGAGAAGCTGGCCTCCCACCTGACCATGATCATCCTGATGGC 
               
               
                   
               
               
                 CAACCTGGCCAGCCAGAAACGGAAGGACGTGCCCGTGAAGCGGAGCACCT 
               
               
                   
               
               
                 TCCTGAAGTACCAGCGGAGCATCAGCAAGACCCTGCAGTACGACAGCAGC 
               
               
                   
               
               
                 ACCAAGACCGTGTCCTTCGAGTACCACCTGAAGGACCCCAGCAAGCTGAT 
               
               
                   
               
               
                 CAAGGGCCTGGAAGATGTGGTGTCCCCCTACAGATTCGTCGTGGGCGTGC 
               
               
                   
               
               
                 ACGAGAAGCCCGACGACGTGATGTCTCACCTGAGCGCCGTGCACATGCGG 
               
               
                   
               
               
                 AAAGAGGCCGGCAGAAAGCGGGACCTGGGCAACAAGATCAACGACGAGAT 
               
               
                   
               
               
                 CACAAAGATCGCCGAGACACAGGAAACCATCTGGGGCTTCGTGGGCAAGA 
               
               
                   
               
               
                 CCATGGACCTGATCGAGGCCAGAACCACCCGGCCTACAACAAAGGCCGCC 
               
               
                   
               
               
                 TACAACCTGCTGCTGCAGGCCACCTTCATGAACTGCTGCAGAGCCGACGA 
               
               
                   
               
               
                 CCTGAAGAACACCGACATCAAGACCTTCGAAGTGATCCCCGACAAGCACC 
               
               
                   
               
               
                 TGGGCCGGATGCTGAGAGCCTTCGTGCCCGAGACAAAGACCGGAACCAGA 
               
               
                   
               
               
                 TTCGTGTACTTCTTCCCATGCAAGGGCAGATGCGACCCCCTGCTGGCCCT 
               
               
                   
               
               
                 GGATTCTTACCTGCAGTGGACCGACCCCATCCCCAAGACCAGAACAACCG 
               
               
                   
               
               
                 ACGAGGACGCCAGATACGACTACCAGCTGCTGCGGAACAGCCTGCTGGGC 
               
               
                   
               
               
                 AGCTACGACGGCTTCATCTCCAAGCAGAGCGACGAGAGCATCTTCAAGAT 
               
               
                   
               
               
                 CCCCAACGGCCCCAAGGCCCACCTGGGCAGACATGTGACAGCCAGCTACC 
               
               
                   
               
               
                 TGAGCAACAACGAGATGGACAAAGAGGCCACCCTGTACGGCAATTGGAGC 
               
               
                   
               
               
                 GCCGCTAGAGAAGAGGGCGTGTCCAGAGTGGCCAAGGCCCGGTACATGCA 
               
               
                   
               
               
                 CACCATCGAGAAGTCCCCCCCCTCCTACCTGTTCGCCTTCCTGAGCGGCT 
               
               
                   
               
               
                 TCTACAACATCACCGCCGAGAGGGCCTGCGAGCTGGTGGACCCCAATAGC 
               
               
                   
               
               
                 AACCCCTGCGAGCAGGACAAGAACATCCCCATGATCAGCGACATCGAGAC 
               
               
                   
               
               
                 ACTGATGGCTCGCTACGGCAAGAACGCCGAGATCATCCCTATGGACGTGC 
               
               
                   
               
               
                 TGGTGTTCCTGAGCAGCTACGCCCGGTTCAAGAACAACGAGGGCAAAGAG 
               
               
                   
               
               
                 TACAAGCTGCAGGCTCGGAGCAGCAGAGGCGTGCCCGACTTCCCCGATAA 
               
               
                   
               
               
                 TGGCAGAACCGCCCTGTACAACGCCCTGACAGCCGCCCACGTGAAGAGGC 
               
               
                   
               
               
                 GGAAGATCAGCATTGTCGTGGGCCGGTCCATCGACACCAGCTGA 
               
               
                   
               
               
                 NLS-PhiC31 (SEQ ID NO: 15): 
               
               
                 atgcctaagaaaaagcggaaagtggatacctacgccggagcctacgacag 
               
               
                   
               
               
                 acagagccgggagagagagaacagcagcgccgccagccccgccacccaga 
               
               
                   
               
               
                 gaagcgccaacgaggataaggccgccgatctgcagagagaggtggagagg 
               
               
                   
               
               
                 gacggcggcagattcagatttgtgggccacttcagcgaggcccctggcac 
               
               
                   
               
               
                 cagcgccttcggcaccgccgagagacccgagttcgagagaatcctgaacg 
               
               
                   
               
               
                 agtgtagggccggcaggctgaacatgatcatcgtgtacgacgtgtcccgg 
               
               
                   
               
               
                 ttcagcaggctgaaggtgatggacgccatccctatcgtgtccgagctgct 
               
               
                   
               
               
                 ggccctgggcgtgaccatcgtgtccacccaggaaggcgtctttagacagg 
               
               
                   
               
               
                 gcaacgtgatggacctgatccacctgatcatgaggctggacgccagccac 
               
               
                   
               
               
                 aaggagagcagcctgaagagcgccaagatcctggacaccaagaacctgca 
               
               
                   
               
               
                 gagggagctgggcggctatgtgggcggcaaggccccctacggcttcgagc 
               
               
                   
               
               
                 tggtgtccgagaccaaggagatcacccggaacggcaggatggtgaacgtg 
               
               
                   
               
               
                 gtgatcaacaagctggcccacagcaccacccccctgaccggccccttcga 
               
               
                   
               
               
                 gtttgagcccgacgtgatcaggtggtggtggcgggagatcaagacccaca 
               
               
                   
               
               
                 agcacctgcctttcaagcccggcagccaggccgccatccaccccggcagc 
               
               
                   
               
               
                 atcaccggcctgtgtaagagaatggacgccgacgccgtgcccaccagagg 
               
               
                   
               
               
                 cgagaccatcggcaagaaaaccgccagcagcgcctgggaccccgccaccg 
               
               
                   
               
               
                 tgatgagaatcctgagggaccctaggatcgccggcttcgccgccgaggtg 
               
               
                   
               
               
                 atctacaagaagaagcccgacggcacccccaccaccaagatcgagggcta 
               
               
                   
               
               
                 cagaatccagagagaccccatcaccctgagacctgtggagctggactgtg 
               
               
                   
               
               
                 gccctatcatcgagcctgccgagtggtacgagctgcaggcctggctggac 
               
               
                   
               
               
                 ggcagaggcagaggcaagggcctgagcagaggccaggccatcctgagcgc 
               
               
                   
               
               
                 catggacaagctgtactgtgagtgtggcgccgtgatgaccagcaagagag 
               
               
                   
               
               
                 gcgaggagagcatcaaggacagctaccggtgccggagaagaaaggtggtg 
               
               
                   
               
               
                 gaccccagcgcccctggccagcacgagggcacctgtaatgtgagcatggc 
               
               
                   
               
               
                 cgccctggacaagttcgtggccgagcggatcttcaacaagatccggcacg 
               
               
                   
               
               
                 ccgagggcgacgaggagaccctggccctgctgtgggaggccgccagaaga 
               
               
                   
               
               
                 ttcggcaagctgaccgaggcccccgagaagagcggcgagagggccaacct 
               
               
                   
               
               
                 ggtggccgagagagccgacgccctgaacgccctggaggagctgtacgagg 
               
               
                   
               
               
                 acagagccgccggagcctatgacggccctgtgggcaggaagcacttcaga 
               
               
                   
               
               
                 aagcagcaggccgccctgaccctgagacagcagggcgccgaggaaagact 
               
               
                   
               
               
                 ggccgagctggaggccgccgaggcccctaagctgcccctggatcagtggt 
               
               
                   
               
               
                 tccccgaggatgccgacgccgaccccaccggccccaagtcctggtggggc 
               
               
                   
               
               
                 agagccagcgtggacgacaagagggtgttcgtgggcctgttcgtggataa 
               
               
                   
               
               
                 gatcgtggtgaccaagagcaccaccggcaggggccagggcacccccatcg 
               
               
                   
               
               
                 agaagagagccagcatcacctgggccaagcctcccaccgacgacgacgag 
               
               
                   
               
               
                 gatgacgcccaggacggcaccgaggacgtggccgcctga 
               
               
                   
               
               
                 NLS-bxb1 (SEQ ID NO: 16): 
               
               
                 ATGGATCCTAAGAAAAAGCGAAAAGTGATGCGAGCCCTGGTGGTCATTCG 
               
               
                   
               
               
                 CCTGAGCAGAGTCACAGACGCTACTACAAGCCCTGAGCGGCAGCTGGAGT 
               
               
                   
               
               
                 CCTGTCAGCAGCTGTGCGCACAGCGAGGATGGGATGTGGTCGGAGTGGCA 
               
               
                   
               
               
                 GAGGATCTGGACGTGAGCGGGGCTGTCGATCCATTCGACCGAAAGCGGAG 
               
               
                   
               
               
                 ACCCAACCTGGCACGATGGCTGGCTTTCGAGGAACAGCCCTTTGATGTGA 
               
               
                   
               
               
                 TCGTCGCCTACAGAGTGGACAGGCTGACACGCTCAATTCGACATCTGCAG 
               
               
                   
               
               
                 CAGCTGGTGCATTGGGCCGAGGATCACAAGAAACTGGTGGTCAGCGCAAC 
               
               
                   
               
               
                 TGAAGCCCACTTCGACACCACAACTCCTTTTGCCGCTGTGGTCATCGCAC 
               
               
                   
               
               
                 TGATGGGCACCGTGGCCCAGATGGAGCTGGAAGCTATCAAGGAGCGAAAC 
               
               
                   
               
               
                 CGGAGCGCAGCCCATTTCAATATTCGGGCCGGGAAATACAGAGGCAGCCT 
               
               
                   
               
               
                 GCCCCCTTGGGGCTATCTGCCTACCCGGGTGGATGGGGAGTGGAGACTGG 
               
               
                   
               
               
                 TGCCAGACCCCGTCCAGAGAGAGAGGATTCTGGAAGTGTACCACAGAGTG 
               
               
                   
               
               
                 GTGGACAACCACGAACCACTGCATCTGGTGGCCCACGATCTGAATAGGCG 
               
               
                   
               
               
                 CGGAGTCCTGTCTCCAAAGGACTATTTTGCTCAGCTGCAGGGAAGGGAGC 
               
               
                   
               
               
                 CACAGGGACGAGAATGGAGTGCTACCGCACTGAAGCGGTCTATGATCAGT 
               
               
                   
               
               
                 GAGGCTATGCTGGGCTATGCAACTCTGAATGGGAAAACCGTGAGAGACGA 
               
               
                   
               
               
                 TGACGGAGCACCACTGGTGCGGGCTGAGCCTATTCTGACAAGAGAGCAGC 
               
               
                   
               
               
                 TGGAAGCTCTGAGGGCAGAACTGGTGAAAACCAGTAGGGCCAAGCCTGCT 
               
               
                   
               
               
                 GTGTCAACACCAAGCCTGCTGCTGCGAGTGCTGTTCTGCGCAGTCTGTGG 
               
               
                   
               
               
                 CGAGCCAGCATACAAATTTGCCGGCGGGGGAAGGAAGCATCCCCGCTATC 
               
               
                   
               
               
                 GATGCCGGAGCATGGGGTTCCCTAAGCACTGTGGAAACGGCACTGTGGCT 
               
               
                   
               
               
                 ATGGCCGAATGGGACGCCTTTTGTGAGGAACAGGTGCTGGATCTGCTGGG 
               
               
                   
               
               
                 GGACGCAGAGCGCCTGGAAAAAGTGTGGGTCGCTGGAAGCGATTCCGCTG 
               
               
                   
               
               
                 TGGAGCTGGCAGAAGTCAATGCCGAGCTGGTGGACCTGACCTCCCTGATC 
               
               
                   
               
               
                 GGATCTCCTGCATACAGGGCAGGCTCCCCACAGCGAGAAGCTCTGGATGC 
               
               
                   
               
               
                 ACGAATTGCTGCACTGGCAGCTCGACAGGAGGAACTGGAGGGGCTGGAAG 
               
               
                   
               
               
                 CCAGACCCTCTGGATGGGAGTGGCGAGAAACAGGCCAGCGGTTTGGGGAT 
               
               
                   
               
               
                 TGGTGGAGGGAGCAGGACACAGCAGCCAAGAACACTTGGCTGAGATCCAT 
               
               
                   
               
               
                 GAATGTCAGGCTGACTTTCGACGTGCGAGGAGGACTGACCCGAACAATCG 
               
               
                   
               
               
                 ATTTTGGCGACCTGCAGGAGTATGAACAGCATCTGCGCCTGGGAAGTGTG 
               
               
                   
               
               
                 GTCGAGCGACTGCACACCGGCATGTCATAA 
               
            
           
         
       
     
     The sequences for some RRS are shown below: 
                    loxP (SEQ ID NO: 17):       ATAACTTCGTATAgcatacatTATACGAAGTTAT               lox2272 (SEQ ID NO: 18):       ATAACTTCGTATAggatacctTATACGAAGTTAT               loxN (SEQ ID NO: 19):       ATAACTTCGTATAgtatacctTATACGAAGTTAT               FRT (SEQ ID NO: 20):       GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC               F3 (SEQ ID NO: 21):       GAAGTTCCTATTCttcaaataGTATAGGAACTTC               F14 (SEQ ID NO: 22):       GAAGTTCCTATTCtatcagaaGTATAGGAACTTC               Rox (SEQ ID NO: 23):       TAACTTTAAATAattggcatTATTTAAAGTTA               VloxP (SEQ ID NO: 24):       TCAATTTCTGAGAactgtcatTCTCGGAAATTGA               Vlox2272 (SEQ ID NO: 25):       TCAATTTCTGAGAagtgtcttTCTCGGAAATTGA               SloxP (SEQ ID NO: 26):       CTCGTGTCCGATAactgtaatTATCGGACATGAT               SloxM1 (SEQ ID NO: 27):       CTCGTGTCCGATAactgtaatTATCGGACACGAG               Slox2272 (SEQ ID NO: 28):       CTCGTGTCCGATAagtgtattTATCGGACATGAT               Vox (SEQ ID NO: 29):       AATAGGTCTGAGAacgcccatTCTCAGACGTATT               B3RT (SEQ ID NO: 30):       GGTTGCTTAAGAATAAGTAATTCTTAAGCAACC               KDRT (SEQ ID NO: 31):       AAACGATATCAGACATTTGTCTGATAATGCTTCATTATCAGACAAATGTC               TGATATCGTTT               B2RT (SEQ ID NO: 32):       GAGTTTCATTAAGGAATAACTAATTCCCTAATGAAACTC               RSRT (SEQ ID NO: 33):       TTGATGAAAGAATAACGTATTCTTTCATCAA               PhiC31 attB (SEQ ID NO: 34):       TGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCC               PhiC31 attP (SEQ ID NO: 35):       GTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGG               Bxb1 attB (SEQ ID NO: 36):       TCGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCCGGGC               Bxb1 attP (SEQ ID NO: 37):       TCGTGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAA               CCC            
Logic Gates
 
     The inventors have constructed a plurality of nucleic acid logic cassettes capable of performing different logic functions. For example,  FIG. 4  depicts an exemplary 6-input-one output genetic device which receives two data inputs, that is controlled by four select inputs S 1 -S 4  to control the output of the GFP expression, where A and B are inputs. This circuit has two data inputs, A and B, and four select inputs, S 1 , S 2 , S 3 , and S 4 . Each select input can control which buffer gates are GFP ON or OFF. Thus, each combination of select inputs configures the device to a specific Boolean logic gate with up to two inputs and one output. Depicted in  FIG. 4  shows A is input from Cre, B is input from Flp, S 1  is input from ϕC31 (phiC31), S 2  is input from Vika, S 3  is input from B3 and S 4  is input from bxb1 recombinases. It should be understood that these are exemplary recombinases for the inputs for the logic gates described herein. The invention contemplates the use of other inputs, which may be chosen by the end-user and used interchangeably with or in place of Cre, or Flp as A and B inputs, or ϕC31 (phiC31), Vika, B3 and bxb1 as S1-S4 recombinases respectively. 
     Sixteen logic gates of the invention (AND, OR, NOT A, NOT B, NOR, NAND, XOR, XNOR, A IMPLY B, B IMPLY A, A NIMPLY B, B NIMPLY A, A, B, FALSE and TRUE) are described below and depicted in  FIGS. 4, 7, and 8A-8B , which depict two-input, 4-input, 6-input and 8-input Boolean logic functions. As shown in  FIG. 7 , logic gates that include “two-input” Boolean logic functions include AND, OR, NOR, NAND, XOR, XNOR, A IMPLY B, B IMPLY A, A NIMPLY B and B NIMPLY A. Thus, a logic gate or a plurality of logic gates is considered to provide all two-input Boolean logic functions if the logic gates or the plurality of logic gates includes AND, OR, NOR, NAND, XOR, XNOR, A IMPLY B, B IMPLY A, A NIMPLY B and B NIMPLY A logic gates. The individual logic gates of the invention, however, are not limited to the genetic circuits depicted in  FIGS. 4, 7, and 8A-8B , but also encompass any of the genetic circuits depicted in all the figures disclosed herein. Any number of the genetic elements in  FIG. 7  may be arranged in a variety of locations and orientations in a given genetic circuit construct of a logic gate to achieve the desired output. For example, alternative genetic circuit constructs for logic gates NOR, AND and XOR are depicted in  FIG. 4  and  FIGS. 7 and 8A-8B . 
     Herein, a promoter is considered to be “operatively linked” when it is in a functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence. A promoter is said to be “conditionally operatively linked” when, upon a genetic recombination event, it is placed in a functional location and orientation in relation to a nucleic acid sequence it regulates. 
     An “inverted” genetic element (e.g., inverted promoter, inverted terminator, inverted target gene) is one that is in the reverse orientation, such that what was the coding (sense) strand is now the non-coding (antisense) strand. In its inverted, reverse orientation, a genetic element is non-functional (e.g., not operatively linked to another genetic element such as a target gene). Function of the genetic element can be restored upon recombination of flanking complementary recognition sites and subsequent inversion of the genetic element back to its correct orientation. Thus, an inverted promoter flanked by recombination recognition sites may be considered to be “conditionally operatively linked” to a downstream target gene if, upon recombination of the flanking complementary recognition sites, the promoter is oriented such that what was the non-coding strand is now the coding strand, and the promoter is able to control transcriptional initiation and/or expression of the target gene. Likewise, an inverted target gene flanked by recombination recognition sites may be “conditionally operatively linked” to an upstream promoter if, upon recombination of the flanking recognition sites, the target gene is oriented such that what was the non-coding strand is now the coding strand, and the upstream promoter is able to control transcriptional initiation and/or expression of the output nucleic acid. 
     Illustrative examples of a promoter operatively linked to a target gene are shown in the NOR, NAND, TRUE, NOT A, NOT B and XNOR logic gates of  FIGS. 4, 7 and 8A-8B . Illustrative examples of a promoter conditionally operatively linked to a target gene are shown in the AND, OR, A, B, A NIMPLY B, B NIMPLY A and XOR logic gates of  FIGS. 4 and 7, 8A-8B . Logic gates A IMPLY B and B IMPLY A contain both a promoter that is operatively linked to a target gene and a promoter that is conditionally operatively linked to a target gene. 
     Herein, a target gene is considered to be downstream of a genetic element if the target gene is located toward the 3′ end and the genetic element is located toward the 5′ end of the coding (sense) strand. One genetic element is considered to be “immediately downstream” of another genetic element the two are proximal to each other (e.g., no other genetic element is located between the two). 
     NOT Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as NOT gates. The simplest Boolean logic gate is referred to as a NOT gate. It takes one input and produces as output its opposite. Disclosed herein are exemplary NOT A and NOT B logic gates in the 6-input-one output genetic device which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, where A and B are inputs ( FIG. 4 ). In  FIG. 4 , where NOT A is used, GFP is expressed when input is received from S 1  and S 3  and input is received from B or when there is no input from either A or B. Similarly, where NOT B is used, GFP is expressed when input is received from S 1  and S 2  and input is received from A or when there is no input from either A or B. 
     One embodiment of the NOT gate is shown in  FIG. 7 . In  FIG. 7 , the NOT gate comprises a promoter and a recombination unit connected to and positioned downstream of the promoter. The recombination unit comprises a pair of RRS specific for a recombinase, each of the RRS flanks each side of a target gene. For a NOT A gate, where A is the recombinase, the target gene is expressed as long as the recombinase is not present; when the recombinase is present, the target gene is excised. 
     AND Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as AND gates. The AND gate is another simple Boolean logic gate. It performs a logical “and” operation on at least two inputs, A and B. Thus, in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is only expressed when inputs A and B are both present. 
     One embodiment of the AND gate is shown in  FIG. 7 , where the AND gate is adapted to receive two inputs. The AND gate comprises a promoter, a U1 connected to and positioned downstream of the promoter, a U2 connected to and positioned downstream of the U1, and a target gene connected to and positioned downstream of the U2. The U1 comprises a first pair of RRS1 specific for a R1, each of the RRS1 flanking each side of a first transcriptional terminator (TT1). The U2 comprises a second pair of RRS2 specific for a R2, each of the RRS2 flanking each side of a second transcriptional terminator (TT2). When both R1 and R2 are present, the TT1 and TT2 are excised, which operatively links the target gene to the promoter. 
     Some embodiments of the AND gates are adapted to receive at least three inputs (e.g., 3, 4, 5, 6, 7, 8, or more) as shown in  FIG. 12B . In these embodiments, the U1, U2 and U3 are connected in series. In the U1, the RRS1 are in an inverse orientation with respect to each other and the first nucleic acid encodes the promoter in an inverted orientation; in the U2, the RRS2 are in the same orientation with respect to each other and the second nucleic acid comprises a transcriptional terminator sequence (TT); and in the U3, the RRS3 are in an inverse orientation with respect to each other and the third nucleic acid comprises a target gene (TG) in an inverted orientation. The RRS1, RRS2, and RRS3 are each recognized by R1, R2, and R3 respectively, and thus only in the presence of the R1, R2 and R3 is the TG operatively linked to the promoter. 
     More recombination units can be added in tandem with the U1, U2, and U3 if more inputs are needed. Accordingly, in some embodiments, the multi-input AND gate further comprises a fourth recombination unit (U4) comprising a fourth pair of recombinase recognition sequences (RRS4) for a fourth recombinase (R4) different from the R1, R2, and R3, the RRS4 being in the same orientation with respect to each other. Each of the RRS4 flanks each side of a fourth nucleic acid sequence comprising a second transcriptional terminator sequence (TT2), and the U4 is positioned in between the U2 and U3. Only in the presence of the R1, R2, R3, and R4 is the TG operatively linked to the promoter. 
     OR Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as OR gates. The OR gate performs a logical “or” operation on input A or input B. Thus, in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is expressed when either inputs A or B, or A and B, are present. 
     One embodiment of the OR gate is shown in  FIG. 7 . The OR gate comprises a promoter, a U1 connected to and positioned downstream of the promoter, a U2 connected to and positioned downstream of the prompter, and a target gene. Each of the RRS1 of the U1 flanks each side of a transcriptional terminator, and each of the RRS2 of the U2 flanks each side of the same transcriptional terminator. The RRS1 are in the same orientation with respect to each other. The RRS2 are in the same orientation with respect to each other. When either the R1 or R2 is present, the transcriptional terminator is excised, thus operatively linking the target gene to the promoter. 
     NOR Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as NOR gates. The NOR gate is a combination of an OR gate with a NOT gate. Thus, in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is only expressed when neither A and B inputs are present. 
     One embodiment of the NOR gate is shown in  FIG. 7 . The NOR gate comprises a promoter, a U1 connected to and positioned downstream of the promoter, a U2 connected to and positioned downstream of the prompter, and a target gene. The U1 comprises the U2. The RRS1 are in the same orientation with respect to each other. The RRS2 are in the same orientation with respect to each other. Each of the RRS2 of the U2 flanks each side of the same target gene. When neither the R1 nor R2 is present, the target gene is expressed. When at least one of the R1 and R2 is present, the target gene is excised. 
     NAND Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as NAND gates. The NAND gate is a combination of an AND gate with a NOT gate. Thus, in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is expressed when inputs A or B are present, or when neither A and B inputs are present. 
     One embodiment of the NAND gate is shown in  FIG. 7 . The NAND gate comprises a promoter, a U1 connected to and positioned downstream of the promoter, and a U2 connected to and positioned downstream of the U1. The U1 comprises a pair of RRS1, wherein each of the RRS1 flanks each side of a TG1, and wherein the RRS1 are in the same orientation as each other. The U2 comprises a pair of RRS2, wherein each of the RRS2 flanks each side of a TG2, and wherein the RRS2 are in the same orientation as each other. The TG1 and TG2 encode the same molecule of interest. There is no or minimal output when both R1 and R2 are present. Under other input conditions, output of similar magnitude is produced. 
     IMPLY Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as A IMPLY B gates (A AND NOT B) and B IMPLY A gates (B AND NOT A). In A IMPLY B gates in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is expressed in all circumstances except when input A is present alone. Similarly, In B IMPLY A gates, GFP is expressed in all circumstances except when input B is present alone. 
     One embodiment of the IMPLY gate is shown in  FIG. 7 . For example, the A IMPLY B gate comprises a promoter, a U1 connected to and positioned downstream of the promoter, and a TG1 connected to and positioned downstream of the U1. The U1 comprises a pair of RRS1 in the same orientation with respect to each other and also a first nucleic acid sequence. Each of the RRS1 flanks each side of the first nucleic acid sequence. The first nucleic acid sequence comprises a U2 and a transcriptional terminator positioned downstream of the U2. The U2 comprises a pair of RRS2 in the same orientation with respect to each other, each of the RRS2 flanking each side of a TG2. The TG1 and TG2 encode the same molecule of interest. When R1 is present and R2 is absent, there is no or minimal output. Under other input conditions, output of similar magnitude is produced. 
     NIMPLY Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as A NIMPLY B and B NIMPLY A gates. In A NIMPLY B gates in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is only expressed when input A is present alone. Similarly, In B NIMPLY A gates, GFP is only expressed when input B is present alone. 
     One embodiment of the NIMPLY gate is shown in  FIG. 7 . For example, the A NIMPLY B gate comprises a promoter and a U1 connected to and positioned downstream of the promoter. The U1 comprises a pair of RRS1 in the same orientation with respect to each other and also a first nucleic acid sequence. Each of the RRS1 flanks each side of the first nucleic acid sequence. The first nucleic acid sequence comprises a U2 and a TG positioned downstream of the U2. The U2 comprises a pair of RRS2 in the same orientation with respect to each other, each of the RRS2 flanking each side of a transcriptional terminator. Output is produced only when R1 is present and R2 is absent. 
     XOR Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as XOR gates. The XOR gate is an “exclusive or” gate. Thus, in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is expressed when either inputs A or input B is present, but not when neither are absent or both A and B inputs are present. 
     One embodiment of the XOR gate is shown in  FIG. 7 . The XOR gate comprises an A NIMPLY B gate and a B NIMPLY A gate. 
     XNOR Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as XNOR gates. The XNOR gate is an “exclusive nor” gate. Thus, in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is expressed when either there are no inputs from both A and B, or when inputs from both A and B are present. 
     One embodiment of the XNOR gate is shown in  FIG. 7 . The XNOR gate comprises a NOR gate and an AND gate. 
     A and B Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as A and B gates. Thus, in an A gate in the LUT 6-input-one output genetic device shown in  FIG. 4  which receives two data inputs, that is controlled by four select inputs S1-S4 to control the output of the GFP expression, GFP is expressed when A input is present, regardless of whether it is alone or whether B input is present. Similarly, in a B gate, GFP is expressed when B input is present, regardless of whether it is alone or whether A input is present. 
     One embodiment of the A gate or B gate is shown in  FIG. 7 . For example, the A gate comprises a promoter, a U1 connected to and positioned downstream of the promoter, and a TG connected to and positioned downstream of the U1. The U1 comprises a pair of RRS1 in the same orientation with respect to each other, wherein each of the RRS1 flanks each side of a transcriptional terminator. Output is produced only when R1 is present. 
     TRUE and FALSE Gates: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as TRUE and FALSE gates. The TRUE gate in  FIG. 4  results in GFP expression at all times. The FALSE gate in  FIG. 4  results in no GFP expression at all times. 
     One embodiment of the TRUE gate or FALSE gate is shown in  FIG. 7 . 
     Half Adder: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as Half Adders. The half adder adds two single binary digits A and B. It has two outputs, sum (S) and carry (C out ). The carry signal represents an overflow into the next digit of a multi-digit addition. The value of the sum is 2C out +S. For example, when the value of input A is 1, the value of input B is 0, the value of the sum is 1; when the value of input A is 1, the value of input B is 1, the value of the sum is 2. As shown in  FIG. 13B , in some embodiments, the half adder can comprise two different reporter genes. In some embodiments, the half adder can comprise one plasmid, two plasmids, or three plasmids. 
     In some embodiments of the half adder being a single transcriptional unit, the first nucleic acid sequence of the U1 of the half adder comprises the U2 and a first target gene (TG1) positioned downstream of the U2. The RRS1 are in the same orientation with respect to each other, and the RRS2 are in the same orientation with respect to each other. The second nucleic acid sequence of the U2 encodes a transcriptional terminator. The U3 has the RRS3 in the same orientation with respect to each other, and the third nucleic acid sequence of the U3 comprises a second TG1. A second target gene (TG2) is positioned downstream of the U3. The RRS2 and RRS3 can be recognized by the same recombinase. 
     Half Subtractor: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as Half Subtractors. The half subtractor is a combinational circuit which is used to perform subtraction of two bits. It has two inputs, A (minuend) and B (subtrahend) and two outputs D (difference) and B out  (borrow). For example, when the value of input A is 1, the value of input B is 0, the value of the difference is 1; when the value of input A is 1, the value of input B is 1, the value of the difference is 0. As shown in  FIG. 13C , in some embodiments, the half subtractor can comprise two different reporter genes. The half subtractor can comprise one plasmid, two plasmids, or three plasmids. 
     In some embodiments of the half subtractor being a single transcriptional unit, the first nucleic acid sequence of the U1 of the half subtractor comprises the U2 and a first target gene (TG1) positioned downstream of the U2. The RRS1 are in the same orientation with respect to each other, and the RRS2 are in the same orientation with respect to each other. The second nucleic acid sequence of the U2 encodes a transcriptional terminator. The U3 has the RRS3 in the same orientation with respect to each other, and the third nucleic acid sequence of the U3 comprises a second TG1 and a second target gene (TG2) separated by ribosomal skip sequences. The RRS2 and RRS3 can be recognized by the same recombinase. 
     Full Adder: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as Full Adders. The full adder adds binary numbers and accounts for values carried in as well as out. A one-bit full adder adds three one-bit numbers, often written as A, B, and C in ; A and B are the operands, and is a bit carried in from the previous less significant stage. The circuit produces a two-bit output, output carry and sum typically represented by the signals C out  and S, where S=2×C out +S. 
       FIG. 5B  shows one embodiment of the full adder. The corresponding genetic architecture is shown in both  FIG. 5B  and  FIG. 16A . The U1 of the full adder has the RRS1 in the same orientation with respect to each other and the first nucleic acid sequence comprises the U2, a fourth recombination unit (U4) positioned downstream of the U2, and a first target gene (TG1) positioned downstream of the U4. The U2 has the RRS2 in the same orientation with respect to each other and the second nucleic acid comprises the U3 and a second target gene (TG2) positioned downstream of the U3. The U3 has the RRS3 in the same orientation with respect to each other and the third nucleic acid comprises a transcriptional terminator sequence. The U4 comprises a fourth pair of recombinase recognition sequences (RRS4) in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a second TG2. The full adder further comprises (i) a fifth recombination unit (U5) positioned downstream of the U1 and comprising a fifth pair of recombinase recognition sequences (RRS5) in the same orientation with respect to each other and a fifth nucleic acid sequence, each of the RRS5 flanking each side of the fifth nucleic acid sequence, the fifth nucleic acid sequence comprising a sixth recombination unit (U6) and a second TG1 positioned downstream of the U6, the U6 comprising a sixth pair of recombinase recognition sequences (RRS6) in the same orientation with respect to each other, wherein each of the RRS6 flanks each side of a third TG2; (ii) a seventh recombination unit (U7) positioned downstream of the U5, the U7 comprising a seventh pair of recombinase recognition sequences (RRS7) in the same orientation with respect to each other, wherein each of the RRS7 flanks each side of a third TG1; and (iii) a fourth TG2 connected to a fourth TG1 and positioned downstream of the U7. In some embodiments, the fourth TG2 is separated from the fourth TG1 by ribosomal skip sequences (2A). In some embodiments, the R1 recognizes the RRS1, the R2 recognizes the RRS2, RRS6, and RRS7, and the R3 recognized the RRS3, RRS4, and RRS5. 
     Full Subtractor: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as Full Subtractors. The full subtractor is a combinational circuit which is used to perform subtraction of three bits. It has three inputs, A (minuend) and B (subtrahend) and C (subtrahend) and two outputs Q (difference) and P (borrow). 
       FIG. 5B  shows one embodiment of the full subtractor. The corresponding genetic architecture is shown in both  FIG. 5B  and  FIG. 16B . The U1 of the full subtractor has the RRS1 in the same orientation with respect to each other and the first nucleic acid sequence comprises the U2, a fourth recombination unit (U4) positioned downstream of the U2, and a first transcriptional terminator sequence (TT1) downstream of the U4. The U2 has the RRS2 in the same orientation with respect to each other and the second nucleic acid comprises the U3 and a first target gene (TG1) positioned downstream of the U3. The U3 has the RRS3 in the same orientation with respect to each other and the third nucleic acid comprises a second transcriptional terminator sequence (TT2). The U4 comprises a fourth pair of recombinase recognition sequences (RRS4) in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a second TG1 and a first TG2 connected together. In some embodiments, the second TG1 is separated from the first TG2 by ribosomal skip sequences (2A). The full subtractor further comprises (i) a fifth recombination unit (U5) positioned downstream of the U1 and comprising a fifth pair of recombinase recognition sequences (RRS5) in the same orientation with respect to each other and a fifth nucleic acid sequence, each of the RRS5 flanking each side of the fifth nucleic acid sequence, the fifth nucleic acid sequence comprising a sixth recombination unit (U6) and a second TG2 positioned downstream of the U6, the U6 comprising a sixth pair of recombinase recognition sequences (RRS6) in the same orientation with respect to each other, wherein each of the RRS6 flanks each side of a third TG1 and a third TG2 connected together; (ii) a seventh recombination unit (U7) positioned downstream of the U5, the U7 comprising a seventh pair of recombinase recognition sequences (RRS7) in the same orientation with respect to each other, wherein each of the RRS7 flanks each side of a third transcriptional terminator sequence (TT3); and (iii) a fourth TG2 connected to a fourth TG1 and positioned downstream of the U7. In some embodiments, the third TG1 is separated from the third TG2 by ribosomal skip sequences (2A). In some embodiments, the fourth TG1 is separated from the fourth TG2 by ribosomal skip sequences (2A). In some embodiments, the R1 recognizes the RRS1, the R2 recognizes the RRS2, RRS6, and RRS7, and the R3 recognized the RRS3, RRS4, and RRS5. 
     Half Adder-Subtractor: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as Half Adder-Subtractors. The half adder-subtractor is a circuit that is capable of adding or subtracting numbers. 
       FIG. 5B  shows one embodiment of the half adder-subtractor. The corresponding genetic architecture is shown in both  FIG. 5B  and  FIG. 16C . The U1 of the half adder-subtractor has the RRS1 in the same orientation with respect to each other and the first nucleic acid sequence comprises the U2, a fourth recombination unit (U4) positioned downstream of the U2, and a first target gene (TG1) positioned downstream of the U4 The U2 has the RRS2 in the same orientation with respect to each other and the second nucleic acid comprises the U3 and a second target gene (TG2) positioned downstream of the U3. The U3 has the RRS3 in the same orientation with respect to each other and the third nucleic acid comprises a first transcriptional terminator sequence (TT1). The U4 comprises a fourth pair of recombinase recognition sequences (RRS4) in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a second TG2. The half adder-subtractor further comprises (i) a fifth recombination unit (U5) positioned downstream of the U1 and comprising a fifth pair of recombinase recognition sequences (RRS5) in the same orientation with respect to each other and a fifth nucleic acid sequence, each of the RRS5 flanking each side of the fifth nucleic acid sequence, the fifth nucleic acid sequence comprising a sixth recombination unit (U6) and a third TG2 and a second TG1 connected together and positioned downstream of the U6, the U6 comprising a sixth pair of recombinase recognition sequences (RRS6) in the same orientation with respect to each other, wherein each of the RRS6 flanks each side of a second transcriptional terminator sequence (TT2); (ii) a seventh recombination unit (U7) positioned downstream of the U5, the U7 comprising a seventh pair of recombinase recognition sequences (RRS7) in the same orientation with respect to each other, wherein each of the RRS7 flanks each side of a fourth TG2; and (iii) a third transcriptional terminator sequence (TT3) positioned downstream of the U7. In some embodiments, the third TG2 is separated from the second TG1 by ribosomal skip sequences (2A). In some embodiments, the R1 recognizes the RRS1, the R2 recognizes the RRS2, RRS6, and RRS7, and the R3 recognized the RRS3, RRS4, and RRS5. 
     Decoder: 
     some embodiments of the nucleic acid logic cassettes are adapted to function as binary decoders. A binary decoder is a combinational logic circuit that converts a binary integer value to an associated pattern of output bits. They are used in a wide variety of applications, including data demultiplexing, seven segment displays, and memory address decoding. Depending on its function, a binary decoder can convert binary information from n input signals to as many as 2′ unique output signals. Some decoders have less than T output lines; in such cases, at least one output pattern will be repeated for different input values. 
     In some embodiments, the decoder described herein is a 2-input 3-output decoder. An exemplary 2-input 3-output decoder is shown in  FIG. 13A . The corresponding genetic architecture is shown in both  FIG. 13A  and  FIG. 17C . In some embodiments, the 2-input 3-output decoder comprises a transcription terminator, a first target gene, a second target gene, and a third target gene connected in series, wherein the transcription terminator is operatively linked to the promoter, whereby (a) when both the R1 and R2 are absent, no output signal is produced; (b) when the R1 is present and the R2 is absent, the first target gene is operatively linked to the promoter, thereby expressing the first target gene to produce a first output signal; (c) when the R1 is absent and the R2 is present, the second target gene is operatively linked to the promoter, thereby expressing the second target gene to produce a second output signal; and (d) when both the R1 and R2 are present, the third target gene is operatively linked to the promoter, thereby expressing the third target gene to produce a third output signal. 
     In some embodiments, the U2 of the 2-input 3-output decoder has the RRS2 in same orientation with respect to each other and the second nucleic acid sequence comprises the U1 upstream of a first target gene (TG1). The TG1 is in the same orientation as the RRS2. And in the U1, each of the RRS1 are in same orientation with respect to each other and the first nucleic acid sequence comprises a transcriptional terminator sequence (TT), wherein the TT is in the same orientation as each of the RRS1. The U3 is located downstream of the U2, and the RRS3 are in the same orientation with respect to each other and the third nucleic acid sequence comprises a second target gene (TG2). The 2-input 3-output decoder optionally comprises a third target gene (TG3) located downstream of the U3. The TG3 is in the same orientation with respect to the RRS3. The RRS1 and RRS3 are recognized by the R1. The operation of the 2-input 3-output decoder adheres to the following rules: (a) absence of the R1 and R2, none of the TG1, TG2 or TG3 are operatively linked to the promoter, or (b) presence of the R1 and absence of the R2 operatively link the promoter to the TG1, or (c) absence of the R1 and presence of the R2 operatively link the promoter to the TG2, or (d) presence of the R1 and R2 operatively links the promoter to the TG3. 
     In some embodiments, the decoder described herein is a 2-input 4-output decoder. In some embodiments, the 2-input 4-output decoder comprises a first target gene (TG1), a second target gene (TG2), a third target gene (TG3), and a fourth target gene (TG4) connected in series, wherein the TG1 is operatively linked to the promoter. Expression of each of the target genes produces a distinct output signal. The operation of the 2-input 4-output decoder adheres to the following rules: (a) when both the R1 and R2 are absent, the TG1 is expressed to produce a first output signal; (b) when the R1 is present and the R2 is absent, the TG2 is operatively linked to the promoter, thereby expressing the TG2 to produce a second output signal; (c) when the R1 is absent and the R2 is present, the TG3 is operatively linked to the promoter, thereby expressing the TG3 to produce a third output signal; and (d) when both the R1 and R2 are present, the TG4 is operatively linked to the promoter, thereby expressing the TG4 to produce a fourth output signal. 
     In some embodiments, an exemplary 2-input 4-output decoder is shown in  FIG. 15B . The corresponding genetic architecture is shown in both  FIG. 15B  and  FIG. 17A . The U2 of the 2-input 4-output decoder has the RRS2 in an inverse orientation with respect to each other and the second nucleic acid sequence comprises the nucleic acid sequences of the U1 and the U3. The U3 is positioned downstream of the U1. And in the U1, the RRS1 are in an inverse orientation with respect to each other and the first nucleic acid sequence comprises a first target gene (TG1) and a second target gene (TG2), wherein the TG1 and the TG2 are in an inverse orientation with respect to each other, and the TG1 is in the same orientation as the promoter. And in the U3, the RRS3 are in an inverse orientation with respect to each other and the third nucleic acid sequence comprises a third target gene (TG3) and a fourth target gene (TG4), wherein the TG3 and the TG4 are in an inverse orientation with respect to each other, and the TG3 is in the same orientation as the promoter. The RRS1 and RRS3 are recognized by the R1. The RRS2 are recognized by the R2. The operation of the 2-input 4-output decoder adheres to the following rules: (a) absence of the R1 and R2 operatively links the promoter to the TG1, or (b) presence of the R1 and absence of the R2 operatively link the promoter to the TG3, or (c) absence of the R1 and presence of the R2 operatively link the promoter to the TG4, or (d) presence of the R1 and R2 operatively links the promoter to the TG2. 
     In some embodiments, an exemplary 2-input 4-output decoder is shown in  FIG. 2B . The corresponding genetic architecture is shown in both  FIG. 2B  and  FIG. 17B . In some embodiments, the RRS2 of the U2 of the 2-input 4-output decoder are in the same orientation with respect to each other and the second nucleic acid sequence comprises the U1 and a first target gene (TG1), wherein the U1 is upstream of the TG1, wherein the TG1 is in the same orientation as the RRS2. And in the U1, the RRS1 are in same orientation with respect to each other and the first nucleic acid sequence encodes a second target gene (TG2), wherein the TG2 is in the same orientation as the RRS1. And the U3 is located downstream of the U2, and the RRS3 are in the same orientation with respect to each other and the third nucleic acid sequence encodes a third target gene (TG3). The 2-input 4-output decoder optionally comprises a fourth target gene (TG4) positioned downstream of the U3. The TG4 is in the same orientation with respect to the RRS3, and the RRS1 and RRS3 are recognized by the R1. The operation of the 2-input 4-output decoder adheres to the following rules: absence of the R1 and R2 operatively links the promoter to the TG2, or presence of the R1 and absence of the R2 operatively link the promoter to the TG1, or absence of the R1 and presence of the R2 operatively link the promoter to the TG3, or presence of the R1 and R2 operatively links the promoter to the TG4. 
     In some embodiments, the decoder described herein is a 3-input 8-output decoder. An embodiment of the 3-input 8-output decoder is shown in  FIG. 14B . The corresponding genetic architecture is shown in both  FIG. 14B  and  FIG. 17D . The 3-input 8-output decoder can comprise a first 2-input 4-output decoder connected with a second 2-input 4-output decoder in series. The first 2-input 4-output has the same genetic architecture as the second 2-input 4-output decoder. The first 2-input 4-output decoder and the second 2-input 4-output decoder are in the same orientation. The 3-input 8-output decoder can further comprise a pair of RRS specific for the R3. Each of the RRS specific for the R3 flanks each side of the two connected 2-input 4-output decoders. The RRS specific for the R3 are in an inverse orientation with respect to each other. And thus when the R3 recognizes the RRS specific for the R3, the nucleic acid sequence comprising the first 2-input 4-output decoder and second 2-input 4-output decoder is inverted with respect to the promoter. As shown in  FIG. 17D , the recombinase that recognizes the RRS2 in the U2 also recognizes the RRS in the U1′ and U3′; the recombinase that recognizes the RRS2′ in the U2′ also recognizes the RRS in the U1 and U3. 
     Another embodiment of the 3-input 8-output decoder is shown in  FIG. 5A . The corresponding genetic architecture is shown in both  FIG. 5A  and  FIG. 17E . The 3-input 8-output decoder comprises (i) a first 2-input 4-output decoder and a second 2-input 4-output decoder connected together and in the same orientation, and (ii) a fourth recombination unit (U4) comprising a fourth pair of recombinase recognition sequence (RRS4) specific for the R3, wherein each of the RRS4 flanks each side of the first 2-input 4-output decoder, wherein the RRS4 are in the same orientation with respect to each other, whereby when R3 recognizes the RRS4, the first nucleic acid logic construct of the 2-input 4-output decoder is excised. As shown in  FIG. 17E , the recombinase that recognizes the RRS2 in the U2 also recognizes the RRS in the U1′ and U3′; the recombinase that recognizes the RRS2′ in the U2′ also recognizes the RRS in the U1 and U3. 
     Promoters 
     In some embodiments, the nucleic acid logic cassettes disclosed herein comprise a promoter sequence. Provided herein are promoter sequences (“promoters”) for use in the recombinase-based synthetic logic and memory systems of the invention. As used herein, a “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof. In some embodiments, the promoter is constitutive. In some embodiments, the promoter is inducible. In some embodiments, the promoter is a mammalian promoter. As discussed herein, a promoter can be applied in any type of cassettes. 
     A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. An “inverted promoter,” as described above, is a promoter in which the nucleic acid sequence is in the reverse orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments of the invention to regulate the state of a logic gate (e.g., high output, “ON,” or low/no output, “OFF”). Thus, in some embodiments, the promoter is an inverted promoter, flanked by complementary recombinase recognition sites that, upon recombination of the sites, inverts to the correct orientation and drives expression of an operatively linked nucleic acid sequence. In some embodiments of the invention, a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence downstream of the promoter. The enhancer may be located at any functional location before or after the promoter and/or the encoded nucleic acid. 
     A promoter is classified as strong or weak according to its affinity for RNA polymerase (and/or sigma factor); this is related to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. The strength of a promoter may depend on whether initiation of transcription occurs at that promoter with high or low frequency. Different promoters with different strengths may be used to construct logic gates with different digitally settable levels of gene output expression (e.g., the level of gene expression initiated from a weak promoter is lower than the level of gene expression initiated from a strong promoter). 
     A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon of a given gene or sequence. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. 
     In some embodiments, a coding nucleic acid segment may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the logic gates disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906). Furthermore, control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts and the like, may be used in accordance with the invention. 
     In some embodiments, the promoter in the cassette or switch is a constitutive or tissue-specific promoter. Tissue-specific promoters are active in a specific type of cells or tissues such as B cells, monocytic cells, leukocytes, macrophages, muscle, pancreatic acinar cells, endothelial cells, astrocytes, and lung. Tissue-specific promoters are available as native or composite promoter. Native promoters, also called minimal promoters, consist of a single fragment from the 5′ region of a given gene. Each of them comprises a core promoter and its natural 5′UTR. In some cases, the 5′UTR contains an intron. Composite promoters combine promoter elements of different origins or were generated by assembling a distal enhancer with a minimal promoter of the same origin. Tissue-specific promoters are commercially available through vendors such as InvivoGen. 
     Non-limiting constitutive promoters include EF1alpha, SFFV, CMV, RSV, SV40, PGK, CAGGS, pTK, Ubc, Ubi, hU6, and H1. 
     In some embodiments, the promoter in the cassette is an inducible promoter. 
     Inducible Promoters 
     As used herein, an “inducible promoter” is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by or contacted by an inducer or inducing agent. An “inducer” or “inducing agent” may be endogenous or a normally exogenous compound or protein that is administered in such a way as to be active in inducing transcriptional activity from the inducible promoter. 
     Inducible promoters for use in accordance with the invention may function in both prokaryotic and eukaryotic host organisms. In some embodiments, mammalian inducible promoters are used. Examples of mammalian inducible promoters for use herein include, without limitation, promoter type P ACT :P AIR , P ART , P BIT , P CR5 , P CTA , P ETR , P NIC , P PIP, PROP , P SPA /P SCA , P TET , P TtgR , promoter type P Rep :P CuO , P ETR , ON8, P NIC , P PIR  ON, P SCA  ON8, P TetO , P UREXS , promoter type P Hyb :teto 7 -ETR 8 -P hcMVmin , tet0 7 -PIR 3 -ETR 8 -P hCMVmin , and scbR 8 -PIR 3 -P hCMVmin . In some embodiments, inducible promoters from other organisms, as well as synthetic promoters designed to function in a prokaryotic or eukaryotic host may be used. Examples of non-mammalian inducible promoters for use herein include, without limitation, Lentivirus promoters {e.g., EFa, CMV, Human Synapsinl (hSynl), CaMKIIa, hGFAP and TPH-2) and Adeno-Associated Virus promoters (e.g., CaMKIIa (AAV5), hSynl (AAV2), hThyl (AAV5), fSST (AAV1), hGFAP (AAV5, AAV8), MBP (AAV8), SST (AAV2)). One important functional characteristic of the inducible promoters of the present invention is their inducibility by exposure to an externally applied inducer. Other examples of inducible promoters include tetracycline inducible (pTRE), streptogramin inducible (pPIR), macrolide inducible (pETR), allolactose or isopropyl β-D-thiogalactopyranoside inducible (pLacO), ponasterone A inducible, coumermycin/novobiocin-regulated gene expression system, hypoxia inducible (hypoxia response elements), TGFbeta inducible (SMAD response elements), amino acid deprivation inducible (ATF3/ATF3/ATF2). More examples of inducible promoters can be found at http://www.sabiosciences.com/reporterassays.php. 
     The administration or removal of an inducer results in a switch between the “ON” or “OFF” states of the transcription of the operatively linked nucleic acid sequence (e.g., nucleic acid encoding a recombinase). Thus, as used herein, the “ON” state of a promoter operatively linked to a nucleic acid sequence refers to the state when the promoter is actively driving transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is expressed). Conversely, the “OFF” state of a promoter operatively linked, or conditionally operatively linked, to a nucleic acid sequence refers to the state when the promoter is not actively driving transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is not expressed). In some embodiments, the inducer can be doxycycline, tamoxifen, rapamycin, or abscisic acid for the promoter operative linked to a nucleic acid sequence encoding a recombinase. 
     An inducible promoter for use in accordance with the invention may be induced by (or repressed by) one or more physiological condition(s), such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s). The extrinsic inducer or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or combinations thereof. The condition(s) and/or agent(s) that induce or repress an inducible promoter can be input(s) of the logic gates described herein. 
     Promoters that are inducible by ionizing radiation can be used in certain embodiments, where gene expression is induced locally in a cell by exposure to ionizing radiation such as UV or x-rays. Radiation inducible promoters include the non-limiting examples of fos promoter, c-jun promoter or at least one CArG domain of an Egr-1 promoter. Further non-limiting examples of inducible promoters include promoters from genes such as cytochrome P450 genes, inducible heat shock protein genes, metallothionein genes, hormone-inducible genes, such as the estrogen gene promoter, and such. In further embodiments, an inducible promoter useful in the methods and systems as described herein can be Zn2+ metallothionein promoter, metallothionein-1 promoter, human metallothionein IIA promoter, lac promoter, lacO promoter, mouse mammary tumor virus early promoter, mouse mammary tumor virus LTR promoter, triose dehydrogenase promoter, herpes simplex virus thymidine kinase promoter, simian virus 40 early promoter or retroviral myeloproliferative sarcoma virus promoter. Examples of inducible promoters also include mammalian probasin promoter, lactalbumin promoter, GRP78 promoter, or the bacterial tetracycline-inducible promoter. Other examples include phorbol ester, adenovirus E1A element, interferon, and serum inducible promoters. 
     In some embodiments, the inducer or inducing agent, i.e., a chemical, a compound or a protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., an inducer can be a transcriptional repressor protein, such as Lad), which itself can be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as a repressor. In other words, in such embodiments, the inducible promoter drives transcription of an operably linked sequence except when the repressor is present. Examples of inducible promoters include but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., the adenovirus late promoter; and the mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin responsive promoters and the like. 
     The promoters for use in the molecular/biological circuits described herein encompass the inducibility of a prokaryotic or eukaryotic promoter by, in part, either of two mechanisms. In some embodiments, the molecular/biological circuits comprise suitable inducible promoters that can be dependent upon transcriptional activators that, in turn, are reliant upon an environmental inducer. In other embodiments, the inducible promoters can be repressed by a transcriptional repressor which itself is rendered inactive by an environmental inducer, such as the product of a sequence driven by another promoter. Thus, unless specified otherwise, an inducible promoter can be either one that is induced by an inducing agent that positively activates a transcriptional activator, or one which is repressed by an inducing agent that negatively regulates a transcriptional repressor. In such embodiments of the various aspects described herein, where it is required to distinguish between an activating and a repressing inducing agent, explicit distinction will be made. 
     Inducible promoters that are useful in the molecular/biological circuits and methods of use described herein also include those controlled by the action of latent transcriptional activators that are subject to induction by the action of environmental inducing agents. Some non-limiting examples include the copper-inducible promoters of the yeast genes CUP1, CRS5, and SOD1 that are subject to copper-dependent activation by the yeast ACE1 transcriptional activator (see e.g. Strain and Culotta, 1996; Hottiger et al., 1994; Lapinskas et al., 1993; and Gralla et al., 1991). Alternatively, the copper inducible promoter of the yeast gene CTT1 (encoding cytosolic catalase T), which operates independently of the ACE1 transcriptional activator (Lapinskas et al., 1993), can be utilized. The copper concentrations required for effective induction of these genes are suitably low so as to be tolerated by most cell systems, including yeast and  Drosophila  cells. Alternatively, other naturally occurring inducible promoters can be used in the present invention including: steroid inducible gene promoters (see e.g. Oligino et al. (1998) Gene Ther. 5: 491-6); galactose inducible promoters from yeast (see e.g. Johnston (1987) Microbiol Rev 51: 458-76; Ruzzi et al. (1987) Mol Cell Biol 7: 991-7); and various heat shock gene promoters. Many eukaryotic transcriptional activators have been shown to function in a broad range of eukaryotic host cells, and so, for example, many of the inducible promoters identified in yeast can be adapted for use in a mammalian host cell as well. For example, a unique synthetic transcriptional induction system for mammalian cells has been developed based upon a GAL4-estrogen receptor fusion protein that induces mammalian promoters containing GAL4 binding sites (Braselmann et al. (1993) Proc Natl Acad Sci USA 90: 1657-61). These and other inducible promoters responsive to transcriptional activators that are dependent upon specific inducers are suitable for use with the cassettes, switches, and methods of use described herein. 
     Inducible promoters useful in some embodiments of the cassettes, switches, and methods of use disclosed herein also include those that are repressed by “transcriptional repressors” that are subject to inactivation by the action of environmental, external agents, or the product of another gene. Such inducible promoters can also be termed “repressible promoters” where it is required to distinguish between other types of promoters in a given module or component of a cassette or switch described herein. Examples include prokaryotic repressors that can transcriptionally repress eukaryotic promoters that have been engineered to incorporate appropriate repressor-binding operator sequences. 
     In some embodiments, repressors for use in the cassettes or switches described herein are sensitive to inactivation by physiologically benign agent. Thus, where a lac repressor protein is used to control the expression of a promoter sequence that has been engineered to contain a lacO operator sequence, treatment of the host cell with IPTG will cause the dissociation of the lac repressor from the engineered promoter containing a lacO operator sequence and allow transcription to occur. Similarly, where a tet repressor is used to control the expression of a promoter sequence that has been engineered to contain a tetO operator sequence, treatment of the host cell with tetracycline or doxycycline will cause the dissociation of the tet repressor from the engineered promoter and allow transcription of the sequence downstream of the engineered promoter to occur. 
     Inducible promoters for use in accordance with the invention include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells). 
     In some embodiments, the inducer used in accordance with the invention is an N-acyl homoserine lactone (AHL), which is a class of signaling molecules involved in bacterial quorum sensing. Quorum sensing is a method of communication between bacteria that enables the coordination of group based behavior based on population density. AHL can diffuse across cell membranes and is stable in growth media over a range of pH values. AHL can bind to transcriptional activators such as LuxR and stimulate transcription from cognate promoters. In some embodiments, the inducer used in accordance with the invention is anhydrotetracycline (aTc), which is a derivative of tetracycline that exhibits no antibiotic activity and is designed for use with tetracycline-controlled gene expression systems, for example, in bacteria. Other inducible promoter systems may be used in accordance with the invention. 
     Inducible promoters useful in the functional modules, cassettes, and switches as described herein for in vivo uses can include those responsive to biologically compatible agents, such as those that are usually encountered in defined animal tissues or cells. An example is the human PAI-1 promoter, which is inducible by tumor necrosis factor. Further suitable examples include cytochrome P450 gene promoters, inducible by various toxins and other agents; heat shock protein genes, inducible by various stresses; hormone-inducible genes, such as the estrogen gene promoter, and such. 
     The administration or removal of an inducer or repressor as disclosed herein results in a switch between the “on” or “off” states of the transcription of the operably linked heterologous target gene. Thus, as defined herein the “on” state, as it refers to a promoter operably linked to a nucleic acid sequence, refers to the state when the promoter is actively driving transcription of the operably linked nucleic acid sequence, i.e., the linked nucleic acid sequence is expressed. Several small molecule ligands have been shown to mediate regulated gene expressions, either in tissue culture cells and/or in transgenic animal models. These include the FK1012 and rapamycin immunosupressive drugs (Spencer et al., 1993; Magari et al., 1997), the progesterone antagonist mifepristone (RU486) (Wang, 1994; Wang et al., 1997), the tetracycline antibiotic derivatives (Gossen and Bujard, 1992; Gossen et al., 1995; Kistner et al., 1996), and the insect steroid hormone ecdysone (No et al., 1996). All of these references are herein incorporated by reference. By way of further example, Yao discloses in U.S. Pat. No. 6,444,871, which is incorporated herein by reference, prokaryotic elements associated with the tetracycline resistance (tet) operon, a system in which the tet repressor protein is fused with polypeptides known to modulate transcription in mammalian cells. The fusion protein is then directed to specific sites by the positioning of the tet operator sequence. For example, the tet repressor has been fused to a transactivator (VP16) and targeted to a tet operator sequence positioned upstream from the promoter of a selected gene (Gussen et al., 1992; Kim et al., 1995; Hennighausen et al., 1995). The tet repressor portion of the fusion protein binds to the operator thereby targeting the VP16 activator to the specific site where the induction of transcription is desired. An alternative approach has been to fuse the tet repressor to the KRAB repressor domain and target this protein to an operator placed several hundred base pairs upstream of a gene. Using this system, it has been found that the chimeric protein, but not the tet repressor alone, is capable of producing a 10 to 15-fold suppression of CMV-regulated gene expression (Deuschle et al., 1995). 
     One example of a repressible promoter useful in the cassettes and switches described herein is the Lac repressor (lacR)/operator/inducer system of  E. coli  that has been used to regulate gene expression by three different approaches: (1) prevention of transcription initiation by properly placed lac operators at promoter sites (Hu and Davidson, 1987; Brown et al., 1987; Figge et al., 1988; Fuerst et al., 1989; Deuschle et al., 1989; (2) blockage of transcribing RNA polymerase II during elongation by a LacR/operator complex (Deuschle et al. (1990); and (3) activation of a promoter responsive to a fusion between LacR and the activation domain of herpes simples virus (HSV) virion protein 16 (VP16) (Labow et al., 1990; Baim et al., 1991). In one version of the Lac system, expression of lac operator-linked sequences is constitutively activated by a LacR-VP16 fusion protein and is turned off in the presence of isopropyl-D-1-thiogalactopyranoside (IPTG) (Labow et al. (1990), cited supra). In another version of the system, a lacR-VP16 variant is used that binds to lac operators in the presence of IPTG, which can be enhanced by increasing the temperature of the cells (Baim et al. (1991), cited supra). 
     Thus, in some embodiments described herein, components of the Lac system are utilized. For example, a lac operator (LacO) can be operably linked to tissue specific promoter, and control the transcription and expression of the heterologous target gene and another protein, such as a repressor protein for another inducible promoter. Accordingly, the expression of the heterologous target gene is inversely regulated as compared to the expression or presence of Lac repressor in the system. 
     Components of the tetracycline (Tc) resistance system of  E. coli  have also been found to function in eukaryotic cells and have been used to regulate gene expression. For example, the Tet repressor (TetR), which binds to tet operator (tetO) sequences in the absence of tetracycline or doxycycline and represses gene transcription, has been expressed in plant cells at sufficiently high concentrations to repress transcription from a promoter containing tet operator sequences (Gatz, C. et al. (1992) Plant J. 2:397-404). In some embodiments described herein, the Tet repressor system is similarly utilized in the molecular/biological circuits described herein. 
     A temperature- or heat-inducible gene regulatory system can also be used in the cassettes and switches described herein, such as the exemplary TIGR system comprising a cold-inducible transactivator in the form of a fusion protein having a heat shock responsive regulator, rheA, fused to the VP16 transactivator (Weber et al. 2003a). The promoter responsive to this fusion thermosensor comprises a rheO element operably linked to a minimal promoter, such as the minimal version of the human cytomegalovirus immediate early promoter. At the permissive temperature of 37° C., the cold-inducible transactivator transactivates the exemplary rheO-CMVmin promoter, permitting expression of the target gene. At 41° C., the cold-inducible transactivator no longer transactivates the rheO promoter. Any such heat-inducible or heat-regulated promoter can be used in accordance with the circuits and methods described herein, including but not limited to a heat-responsive element in a heat shock gene (e.g., hsp20-30, hsp27, hsp40, hsp60, hsp70, and hsp90). See Easton et al. (2000) Cell Stress Chaperones 5(4):276-290; Csermely et al. (1998) Pharmacol Ther 79(2): 129-1 68; Ohtsuka &amp; Hata (2000) Int J Hyperthermia 16(3):231-245; and references cited therein. Sequence similarity to heat shock proteins and heat-responsive promoter elements have also been recognized in genes initially characterized with respect to other functions, and the DNA sequences that confer heat inducibility are suitable for use in the disclosed gene therapy vectors. For example, expression of glucose-responsive genes (e.g., grp94, grp78, mortalin/grp75) (Merrick et al. (1997) Cancer Lett 119(2): 185-1 90; Kiang et al. (1998) FASEB J 12(14):1571-16-579), calreticulin (Szewczenko-Pawlikowski et al. (1997) MoI Cell Biochem 177(1-2): 145-1 52); clusterin (Viard et al. (1999) J Invest Dermatol 112(3):290-296; Michel et al. (1997) Biochem J 328(Pt1):45-50; Clark &amp; Griswold (1997) J Androl 18(3):257-263), histocompatibility class I gene (HLA-G) (Ibrahim et al. (2000) Cell Stress Chaperones 5(3):207-218), and the Kunitz protease isoform of amyloid precursor protein (Shepherd et al. (2000) Neuroscience 99(2):31 7-325) are upregulated in response to heat. In the case of clusterin, a 14 base pair element that is sufficient for heat-inducibility has been delineated (Michel et al. (1997) Biochem J 328(Pt1):45-50). Similarly, a two sequence unit comprising a 10- and a 14-base pair element in the calreticulin promoter region has been shown to confer heat-inducibility (Szewczenko-Pawlikowski et al. (1997) MoI Cell Biochem 177(1-2): 145-1 52). 
     Other inducible promoters useful in the cassettes and switches described herein include the erythromycin-resistance regulon from  E. coli , having repressible (Eoff) and inducible (Eon) systems responsive to macrolide antibiotics, such as erythromycin, clarithromycin, and roxithromycin (Weber et al., 2002). The Eoff system utilizes an erythromycin-dependent transactivator, wherein providing a macrolide antibiotic represses transgene expression. In the Eon system, the binding of the repressor to the operator results in repression of transgene expression. Thus, in the presence of macrolides, gene expression is induced. 
     Fussenegger et al. (2000) describe repressible and inducible systems using a Pip (pristinamycin-induced protein) repressor encoded by the streptogramin resistance operon of  Streptomyces coelicolor , wherein the systems are responsive to streptogramin-type antibiotics (such as, for example, pristinamycin, virginiamycin, and Synercid). The Pip DNA-binding domain is fused to a VP16 transactivation domain or to the KRAB silencing domain, for example. The presence or absence of, for example, pristinamycin, regulates the PipON and PipOFF systems in their respective manners, as described therein. 
     Another example of a promoter expression system useful for the cassettes and switches described herein utilizes a quorum-sensing (referring to particular prokaryotic molecule communication systems having diffusible signal molecules that prevent binding of a repressor to an operator site, resulting in repression of a target regulon) system. For example, Weber et al. (2003b) employ a fusion protein comprising the  Streptomyces coelicolor  quorum-sending receptor to a transactivating domain that regulates a chimeric promoter having a respective operator that the fusion protein binds. The expression is fine-tuned with non-toxic butyrolactones, such as SCB1 and MP133. 
     In some embodiments, multiregulated, multigene gene expression systems that are functionally compatible with one another are utilized in the modules, cassettes, and switches described herein (see, for example, Kramer et al. (2003)). For example, in Weber et al. (2002), the macrolide-responsive erythromycin resistance regulon system is used in conjunction with a streptogramin (PIP)-regulated and tetracycline-regulated expression systems. 
     Other promoters responsive to non-heat stimuli can also be used. For example, the mortalin promoter is induced by low doses of ionizing radiation (Sadekova (1997) Int J Radiat Biol 72(6):653-660), the hsp27 promoter is activated by 17-β-estradiol and estrogen receptor agonists (Porter et al. (2001) J MoI Endocrinol 26(1):31-42), the HLA-G promoter is induced by arsenite, hsp promoters can be activated by photodynamic therapy (Luna et al. (2000) Cancer Res 60(6): 1637-1 644). A suitable promoter can incorporate factors such as tissue-specific activation. For example, hsp70 is transcriptionally impaired in stressed neuroblastoma cells (Drujan &amp; De Maio (1999) 12(6):443-448) and the mortalin promoter is up-regulated in human brain tumors (Takano et al. (1997) Exp Cell Res 237(1):38-45). A promoter employed in methods described herein can show selective up-regulation in tumor cells as described, for example, for mortalin (Takano et al. (1997) Exp Cell Res 237(1):38-45), hsp27 and calreticulin (Szewczenko-Pawlikowski et al. (1997) MoI Cell Biochem 177(1-2): 145-1 52; Yu et al. (2000) Electrophoresis 2 1(14):3058-3068)), grp94 and grp78 (Gazit et al. (1999) Breast Cancer Res Treat 54(2): 135-146), and hsp27, hsp70, hsp73, and hsp90 (Cardillo et al. (2000) Anticancer Res 20(6B):4579-4583; Strik et al. (2000) Anticancer Res 20(6B):4457-4552). 
     In some exemplary embodiments, an inducible promoter is an arabinose-inducible promoter P BAD  comprising the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 38) 
               
            
           
           
               
            
               
                 AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT 
               
               
                   
               
               
                 TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCAT 
               
               
                   
               
               
                 TCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTG 
               
               
                   
               
               
                 TCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACA 
               
               
                   
               
               
                 CTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTG 
               
               
                   
               
               
                 ACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATA. 
               
            
           
         
       
     
     In some exemplary embodiments, an inducible promoter is a LuxR-inducible promoter P LUXR  comprising the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 39) 
               
            
           
           
               
            
               
                 ACCTGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCG 
               
               
                   
               
               
                 AATAAA. 
               
            
           
         
       
     
     In some exemplary embodiments, an inducible promoter is a mutated LuxR-targeted promoter with modulated binding efficiency for LuxR, such as, for example, pluxR3: 
                    (SEQ ID NO: 40)                 AATTTGGGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGA               ATAAA;            
pluxR28:
 
                    (SEQ ID NO: 41)                 CTGGCGGGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGA               ATAAA;            
pluxR56:
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 42) 
               
            
           
           
               
            
               
                 TGGGGTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGA 
               
               
                   
               
               
                 ATAAA. 
               
            
           
         
       
     
     In some exemplary embodiments, the inducible promoter comprises an Anhydrotetracycline (aTc)-inducible promoter as provided in PLtetO-1 (Pubmed Nucleotide #U66309) with the sequence comprising: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 43) 
               
            
           
           
               
            
               
                 GCATGCTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGA 
               
               
                   
               
               
                 TACTGAGCACATCAGCAGGACGCACTGACCAGGA. 
               
            
           
         
       
     
     In some exemplary embodiments, the inducible promoter is an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter. In one embodiment, the IPTG-inducible promoter comprises the P TAC  sequence found in the vector encoded by PubMed Accession ID #EU546824. In one embodiment, the IPTG-inducible promoter sequence comprises the P Trc-2  sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 44) 
               
            
           
           
               
            
               
                 CCATCGAATGGCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTAT 
               
               
                   
               
               
                 AATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGA. 
               
            
           
         
       
     
     In some exemplary embodiments, the IPTG-inducible promoter comprises the P LlacO-1  sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 45) 
               
            
           
           
               
            
               
                 ATAAATGTGAGCGGATAACATTGACATTGTGAGCGGATAACAAGATACTG 
               
               
                   
               
               
                 AGCACTCAGCAGGACGCACTGACC. 
               
            
           
         
       
     
     In some exemplary embodiments, the IPTG-inducible promoter comprises the P AllacO-1  sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 46) 
               
            
           
           
               
            
               
                 AAAATTTATCAAAAAGAGTGTTGACTTGTGAGCGGATAACAATGATACTT 
               
               
                   
               
               
                 AGATTCAATTGTGAGCGGATAACAATTTCACACA. 
               
            
           
         
       
     
     In some exemplary embodiments, the IPTG-inducible promoter comprises the P lac/ara-1  sequence 
     
       
         
           
               
            
               
                 (SEQ ID NO: 47) 
               
            
           
           
               
            
               
                 CATAGCATTTTTATCCATAAGATTAGCGGATCCTAAGCTTTACAATTGTG 
               
               
                   
               
               
                 AGCGCTCACAATTATGATAGATTCAATTGTGAGCGGATAACAATTTCAC 
               
               
                   
               
               
                 ACA. 
               
            
           
         
       
     
     In some exemplary embodiments, the inducible promoter sequence comprises the P Ls1com  sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 48) 
               
            
           
           
               
            
               
                 GCATGCACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGA 
               
               
                   
               
               
                 CATAAATACCACTGGCGGTtATAaTGAGCACATCAGCAGG//GTATGCAA 
               
               
                   
               
               
                 AGGA. 
               
            
           
         
       
     
     Other non-limiting examples of promoters are provided in Tables 1-36. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Examples of Constitutive  E. coli  σ 70  Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I14018 
                 SEQ ID NO: 49 P(Bla) 
                 ... 
               
               
                   
                   
                 gtttatacataggcgagtactctgttatgg 
               
               
                   
               
               
                 BBa_I14033 
                 SEQ ID NO: 50 P(Cat) 
                 ... 
               
               
                   
                   
                 agaggttccaactttcaccataatgaaaca 
               
               
                   
               
               
                 BBa_I14034 
                 SEQ ID NO: 51 P(Kat) 
                 ... 
               
               
                   
                   
                 taaacaactaacggacaattctacctaaca 
               
               
                   
               
               
                 BBa_I732021 
                 SEQ ID NO: 52 Template for Building Primer Family 
                 ... 
               
               
                   
                 Member 
                 acatcaagccaaattaaacaggattaacac 
               
               
                   
               
               
                 BBa_I742126 
                 SEQ ID NO: 53 Reverse lambda cI-regulated promoter 
                 ... 
               
               
                   
                   
                 gaggtaaaatagtcaacacgcacggtgtta 
               
               
                   
               
               
                 BBa_J01006 
                 SEQ ID NO: 54 Key Promoter absorbs 3 
                 ... 
               
               
                   
                   
                 caggccggaataactccctataatgcgcca 
               
               
                   
               
               
                 BBa_J23100 
                 SEQ ID NO: 55 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtacagtgctagc 
               
               
                   
               
               
                 BBa_J23101 
                 SEQ ID NO: 56 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctaggtattatgctagc 
               
               
                   
               
               
                 BBa_J23102 
                 SEQ ID NO: 57 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctaggtactgtgctagc 
               
               
                   
               
               
                 BBa_J23103 
                 SEQ ID NO: 58 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctagggattatgctagc 
               
               
                   
               
               
                 BBa_J23104 
                 SEQ ID NO: 59 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctaggtattgtgctagc 
               
               
                   
               
               
                 BBa_J23105 
                 SEQ ID NO: 60 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtactatgctagc 
               
               
                   
               
               
                 BBa_J23106 
                 SEQ ID NO: 61 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtatagtgctagc 
               
               
                   
               
               
                 BBa_J23107 
                 SEQ ID NO: 62 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagccctaggtattatgctagc 
               
               
                   
               
               
                 BBa_J23108 
                 SEQ ID NO: 63 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctaggtataatgctagc 
               
               
                   
               
               
                 BBa_J23109 
                 SEQ ID NO: 64 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctagggactgtgctagc 
               
               
                   
               
               
                 BBa_J23110 
                 SEQ ID NO: 65 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtacaatgctagc 
               
               
                   
               
               
                 BBa_J23111 
                 SEQ ID NO: 66 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtatagtgctagc 
               
               
                   
               
               
                 BBa_J23112 
                 SEQ ID NO: 67 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctagggattatgctagc 
               
               
                   
               
               
                 BBa_J23113 
                 SEQ ID NO: 68 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctagggattatgctagc 
               
               
                   
               
               
                 BBa_J23114 
                 SEQ ID NO: 69 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtacaatgctagc 
               
               
                   
               
               
                 BBa_J23115 
                 SEQ ID NO: 70 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagcccttggtacaatgctagc 
               
               
                   
               
               
                 BBa_J23116 
                 SEQ ID NO: 71 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctagggactatgctagc 
               
               
                   
               
               
                 BBa_J23117 
                 SEQ ID NO: 72 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctagggattgtgctagc 
               
               
                   
               
               
                 BBa_J23118 
                 SEQ ID NO: 73 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 ggctagctcagtectaggtattgtgctagc 
               
               
                   
               
               
                 BBa_J23119 
                 SEQ ID NO: 74 constitutive promoter family member 
                 ... 
               
               
                   
                   
                 agctagctcagtcctaggtataatgctagc 
               
               
                   
               
               
                 BBa_J23150 
                 SEQ ID NO: 75 1bp mutant from J23107 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtattatgctagc 
               
               
                   
               
               
                 BBa_J23151 
                 SEQ ID NO: 76 1bp mutant from J23114 
                 ... 
               
               
                   
                   
                 ggctagctcagtcctaggtacaatgctagc 
               
               
                   
               
               
                 BBa_J44002 
                 SEQ ID NO: 77 pBAD reverse 
                 ... 
               
               
                   
                   
                 aaagtgtgacgccgtgcaaataatcaatgt 
               
               
                   
               
               
                 BBa_J48104 
                 SEQ ID NO: 78 NikR promoter, a protein of the ribbon 
                 ...gacgaatacttaaaatcgtcatacttattt 
               
               
                   
                 helix-helix family of transcription factors that repress 
               
               
                   
                 expre 
               
               
                   
               
               
                 BBa_J54200 
                 SEQ ID NO: 79 lacq_Promoter 
                 ... 
               
               
                   
                   
                 aaacctttcgcggtatggcatgatagcgcc 
               
               
                   
               
               
                 BBa_J56015 
                 SEQ ID NO: 80 lacIQ—promoter sequence 
                 ... 
               
               
                   
                   
                 tgatagcgcccggaagagagtcaattcagg 
               
               
                   
               
               
                 BBa_J64951 
                 SEQ ID NO: 81  E. coli  CreABCD phosphate sensing 
                 ...ttatttaccgtgacgaactaattgctcgtg 
               
               
                   
                 operon promoter 
               
               
                   
               
               
                 BBa_K088007 
                 SEQ ID NO: 82 GlnRS promoter 
                 ...catacgccgttatacgttgtttacgctttg 
               
               
                   
               
               
                 BBa_K119000 
                 SEQ ID NO: 83 Constitutive weak promoter of lacZ 
                 ...ttatgcttccggctcgtatgttgtgtggac 
               
               
                   
               
               
                 BBa_K119001 
                 SEQ ID NO: 84 Mutated LacZ promoter 
                 ... 
               
               
                   
                   
                 ttatgcttccggctcgtatggtgtgtggac 
               
               
                   
               
               
                 BBa_K137029 
                 SEQ ID NO: 85 constitutive promoter with (TA)10 
                 ...atatatatatatatataatggaagcgtttt 
               
               
                   
                 between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137030 
                 SEQ ID NO: 86 constitutive promoter with (TA)9 
                 ...atatatatatatatataatggaagcgtttt 
               
               
                   
                 between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137031 
                 SEQ ID NO: 87 constitutive promoter with (C)10 
                 ... 
               
               
                   
                 between −10 and −35 elements 
                 ccccgaaagcttaagaatataattgtaagc 
               
               
                   
               
               
                 BBa_K137032 
                 SEQ ID NO: 88 constitutive promoter with (C)12 
                 ... 
               
               
                   
                 between −10 and −35 elements 
                 ccccgaaagcttaagaatataattgtaagc 
               
               
                   
               
               
                 BBa_K137085 
                 SEQ ID NO: 89 optimized (TA) repeat constitutive 
                 ...tgacaatatatatatatatataatgctagc 
               
               
                   
                 promoter with 13 bp between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137086 
                 SEQ ID NO: 90 optimized (TA) repeat constitutive 
                 ...acaatatatatatatatatataatgctagc 
               
               
                   
                 promoter with 15 bp between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137087 
                 SEQ ID NO: 91 optimized (TA) repeat constitutive 
                 ...aatatatatatatatatatataatgctagc 
               
               
                   
                 promoter with 17 bp between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137088 
                 SEQ ID NO: 92 optimized (TA) repeat constitutive 
                 ...tatatatatatatatatatataatgctagc 
               
               
                   
                 promoter with 19 bp between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137089 
                 SEQ ID NO: 93 optimized (TA) repeat constitutive 
                 ...tatatatatatatatatatataatgctagc 
               
               
                   
                 promoter with 21 bp between −10 and −35 elements 
               
               
                   
               
               
                 BBa_K137090 
                 SEQ ID NO: 94 optimized (A) repeat constitutive 
                 ... 
               
               
                   
                 promoter with 17 bp between −10 and −35 elements 
                 aaaaaaaaaaaaaaaaaatataatgctagc 
               
               
                   
               
               
                 BBa_K137091 
                 SEQ ID NO: 95 optimized (A) repeat constitutive 
                 ... 
               
               
                   
                 promoter with 18 bp between −10 and −35 elements 
                 aaaaaaaaaaaaaaaaaatataatgctagc 
               
               
                   
               
               
                 BBa_K256002 
                 SEQ ID NO: 96 J23101:GFP 
                 ... 
               
               
                   
                   
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K256018 
                 SEQ ID NO: 97 J23119:IFP 
                 ... 
               
               
                   
                   
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K256020 
                 SEQ ID NO: 98 J23119:HO1 
                 ... 
               
               
                   
                   
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K256033 
                 SEQ ID NO: 99 Infrared signal reporter 
                 ... 
               
               
                   
                 (J23119:IFP:J23119:HO1) 
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K292000 
                 SEQ ID NO: 100 Double terminator + 
                 ... 
               
               
                   
                 constitutive promoter 
                 ggctagctcagtcctaggtacagtgctagc 
               
               
                   
               
               
                 BBa_K292001 
                 SEQ ID NO: 101 Double terminator + 
                 ... 
               
               
                   
                 Constitutive promoter + Strong RBS 
                 tgctagctactagagattaaagaggagaaa 
               
               
                   
               
               
                 BBa_M13101 
                 SEQ ID NO: 102 M13K07 gene I promoter 
                 ...cctgtttttatgttattctctctgtaaagg 
               
               
                   
               
               
                 BBa_M13102 
                 SEQ ID NO: 103 M13K07 gene II promoter 
                 ...aaatatttgcttatacaatcttcctgtttt 
               
               
                   
               
               
                 BBa_M13103 
                 SEQ ID NO: 104 M13K07 gene III promoter 
                 ... 
               
               
                   
                   
                 gctgataaaccgatacaattaaaggctcct 
               
               
                   
               
               
                 BBa_M13104 
                 SEQ ID NO: 105 M13K07 gene IV promoter 
                 ...ctcttctcagcgtcttaatctaagctatcg 
               
               
                   
               
               
                 BBa_M13105 
                 SEQ ID NO: 106 M13K07 gene V promoter 
                 ... 
               
               
                   
                   
                 atgagccagttcttaaaatcgcataaggta 
               
               
                   
               
               
                 BBa_M13106 
                 SEQ ID NO: 107 M13K07 gene VI promoter 
                 ...ctattgattgtgacaaaataaacttattcc 
               
               
                   
               
               
                 BBa_M13108 
                 SEQ ID NO: 108 M13K07 gene VIII promoter 
                 ... 
               
               
                   
                   
                 gtttcgcgcttggtataatcgctgggggtc 
               
               
                   
               
               
                 BBa_M13110 
                 SEQ ID NO: 109 M13110 
                 ...ctttgcttctgactataatagtcagggtaa 
               
               
                   
               
               
                 BBa_M31519 
                 SEQ ID NO: 110 Modified promoter sequence 
                 ... 
               
               
                   
                 of g3. 
                 aaaccgatacaattaaaggctcctgctagc 
               
               
                   
               
               
                 BBa_R1074 
                 SEQ ID NO: 111 Constitutive Promoter I 
                 ... 
               
               
                   
                   
                 gccggaataactccctataatgcgccacca 
               
               
                   
               
               
                 BBa_R1075 
                 SEQ ID NO: 112 Constitutive Promoter II 
                 ... 
               
               
                   
                   
                 gccggaataactccctataatgcgccacca 
               
               
                   
               
               
                 BBa_S03331 
                 SEQ ID NO: 113 
                 ttgacaagcttttcctcagctccgtaaact 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Examples of Constitutive  E. coli  σ 70  Promoters 
               
            
           
           
               
               
               
            
               
                 Identifier 
                   
                 Sequence 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 BBa_J23119 
                 SEQ ID NO: 114 
                 ttgacagctagctcagtcctaggtataatgctagc 
                 n/a 
               
               
                   
               
               
                 BBa_J23100 
                 SEQ ID NO: 115 
                 ttgacggctagctcagtcctaggtacagtgctagc 
                 1 
               
               
                   
               
               
                 BBa_J23101 
                 SEQ ID NO: 116 
                 tttacagctagctcagtcctaggtattatgctagc 
                 0.70 
               
               
                   
               
               
                 BBa_J23102 
                 SEQ ID NO: 117 
                 ttgacagctagctcagtcctaggtactgtgctagc 
                 0.86 
               
               
                   
               
               
                 BBa_J23103 
                 SEQ ID NO: 118 
                 ctgatagctagctcagtcctagggattatgctagc 
                 0.01 
               
               
                   
               
               
                 BBa_J23104 
                 SEQ ID NO: 119 
                 ttgacagctagctcagtcctaggtattgtgctagc 
                 0.72 
               
               
                   
               
               
                 BBa_J23105 
                 SEQ ID NO: 120 
                 tttacggctagctcagtcctaggtactatgctagc 
                 0.24 
               
               
                   
               
               
                 BBa_J23106 
                 SEQ ID NO: 121 
                 tttacggctagctcagtcctaggtatagtgctagc 
                 0.47 
               
               
                   
               
               
                 BBa_J23107 
                 SEQ ID NO: 122 
                 tttacggctagctcagccctaggtattatgctagc 
                 0.36 
               
               
                   
               
               
                 BBa_J23108 
                 SEQ ID NO: 123 
                 ctgacagctagctcagtcctaggtataatgctagc 
                 0.51 
               
               
                   
               
               
                 BBa_J23109 
                 SEQ ID NO: 124 
                 tttacagctagctcagtcctagggactgtgctagc 
                 0.04 
               
               
                   
               
               
                 BBa_J23110 
                 SEQ ID NO: 125 
                 tttacggctagctcagtcctaggtacaatgctagc 
                 0.33 
               
               
                   
               
               
                 BBa_J23111 
                 SEQ ID NO: 126 
                 ttgacggctagctcagtcctaggtatagtgctagc 
                 0.58 
               
               
                   
               
               
                 BBa_J23112 
                 SEQ ID NO: 127 
                 ctgatagctagctcagtcctagggattatgctagc 
                 0.00 
               
               
                   
               
               
                 BBa_J23113 
                 SEQ ID NO: 128 
                 ctgatggctagctcagtcctagggattatgctagc 
                 0.01 
               
               
                   
               
               
                 BBa_J23114 
                 SEQ ID NO: 129 
                 tttatggctagctcagtcctaggtacaatgctagc 
                 0.10 
               
               
                   
               
               
                 BBa_J23115 
                 SEQ ID NO: 130 
                 tttatagctagctcagcccttggtacaatgctagc 
                 0.15 
               
               
                   
               
               
                 BBa_J23116 
                 SEQ ID NO: 131 
                 ttgacagctagctcagtcctagggactatgctagc 
                 0.16 
               
               
                   
               
               
                 BBa_J23117 
                 SEQ ID NO: 132 
                 ttgacagctagctcagtcctagggattgtgctagc 
                 0.06 
               
               
                   
               
               
                 BBa_J23118 
                 SEQ ID NO: 133 
                 ttgacggctagctcagtcctaggtattgtgctagc 
                 0.56 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Examples of Constitutive  E. coli  σ S  Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J45992 
                 SEQ ID NO: 134 Full-length stationary phase osmY 
                 ... 
               
               
                   
                 promoter 
                 ggtttcaaaattgtgatctatatttaacaa 
               
               
                   
               
               
                 BBa_J45993 
                 SEQ ID NO: 135 Minimal stationary phase osmY 
                 ... 
               
               
                   
                 promoter 
                 ggtttcaaaattgtgatctatatttaacaa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Examples of Constitutive  E. coli  σ 32  Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J45504 
                 SEQ ID NO: 136 htpG 
                 ... 
               
               
                   
                 Heat Shock Promoter 
                 tctattccaataaagaaat 
               
               
                   
                   
                 cttcctgcgtg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Examples of Constitutive  B. subtilis  σ A  Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K143012 
                 SEQ ID NO: 137 Promoter veg a 
                 ... 
               
               
                   
                 constitutive promoter for  B. subtilis   
                 aaaaatgggctcgtgttgtacaataaatgt 
               
               
                   
               
               
                 BBa_K143013 
                 SEQ ID NO: 138 Promoter 43 a 
                 ... 
               
               
                   
                 constitutive promoter for  B. subtilis   
                 aaaaaaagcgcgcgattatgtaaaatataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Examples of Constitutive  B. subtilis  σ B  Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K143010 
                 SEQ ID NO: 139 Promoter etc for  B. subtilis   
                 ...atccttatcgttatgggtattgtttgtaat 
               
               
                   
               
               
                 BBa_K143011 
                 SEQ ID NO: 140 Promoter gsiB for  B. subtilis   
                 ... 
               
               
                   
                   
                 taaaagaattgtgagcgggaatacaacaac 
               
               
                   
               
               
                 BBa_K143013 
                 SEQ ID NO: 141 Promoter 43 a constitutive 
                 ... 
               
               
                   
                 promoter for  B. subtilis   
                 aaaaaaagcgcgcgattatgtaaaatataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Examples of Constitutive Promoters from Miscellaneous Prokaryotes 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K112706 
                 SEQ ID NO: 142 Pspv2 from  Salmonella   
                 ...tacaaaataattcccctgcaaacattatca 
               
               
                   
               
               
                 BBa_K112707 
                 SEQ ID NO: 143 Pspv from  Salmonella   
                 ...tacaaaataattcccctgcaaacattatcg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Examples of Constitutive Promoters from bacteriophage T7 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I712074 
                 SEQ ID NO: 144 T7 promoter (strong 
                 ...agggaatacaagctacttgttctttttgca 
               
               
                   
                 promoter from T7 bacteriophage) 
               
               
                   
               
               
                 BBa_I719005 
                 SEQ ID NO: 145 T7 Promoter 
                 taatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_J34814 
                 SEQ ID NO: 146 T7 Promoter 
                 gaatttaatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_J64997 
                 SEQ ID NO: 147 T7 consensus −10 and rest 
                 taatacgactcactatagg 
               
               
                   
               
               
                 BBa_K113010 
                 SEQ ID NO: 148 overlapping T7 promoter 
                 ... 
               
               
                   
                   
                 gagtcgtattaatacgactcactatagggg 
               
               
                   
               
               
                 BBa_K113011 
                 SEQ ID NO: 149 more overlapping T7 
                 ... 
               
               
                   
                 promoter 
                 agtgagtcgtactacgactcactatagggg 
               
               
                   
               
               
                 BBa_K113012 
                 SEQ ID NO: 150 weaken overlapping T7 
                 ... 
               
               
                   
                 promoter 
                 gagtcgtattaatacgactctctatagggg 
               
               
                   
               
               
                 BBa_R0085 
                 SEQ ID NO: 151 T7 Consensus Promoter 
                 taatacgactcactatagggaga 
               
               
                   
                 Sequence 
               
               
                   
               
               
                 BBa_R0180 
                 SEQ ID NO: 152 T7 RNAP promoter 
                 ttatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_R0181 
                 SEQ ID NO: 153 T7 RNAP promoter 
                 gaatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_R0182 
                 SEQ ID NO: 154 T7 RNAP promoter 
                 taatacgtctcactatagggaga 
               
               
                   
               
               
                 BBa_R0183 
                 SEQ ID NO: 155 T7 RNAP promoter 
                 tcatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_Z0251 
                 SEQ ID NO: 156 T7 strong promoter 
                 ... 
               
               
                   
                   
                 taatacgactcactatagggagaccacaac 
               
               
                   
               
               
                 BBa_Z0252 
                 SEQ ID NO: 157 T7 weak binding and 
                 ... 
               
               
                   
                 processivity 
                 taattgaactcactaaagggagaccacagc 
               
               
                   
               
               
                 BBa_Z0253 
                 SEQ ID NO: 158 T7 weak binding promoter 
                 ... 
               
               
                   
                   
                 cgaagtaatacgactcactattagggaaga 
               
               
                   
               
               
                   
                 SEQ ID NO: 159 T7 14.3 m 
                 attaaccctcactaaagggaga 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Examples of Constitutive Promoters from 
               
               
                 bacteriophage SP6 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J64998 
                 SEQ ID NO: 160 
                 atttaggtgacactataga 
               
               
                   
                 consensus −10 and 
               
               
                   
                 rest from SP6 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Examples of Constitutive Promoters from Yeast 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I766555 
                 SEQ ID NO: 161 pCyc (Medium) Promoter 
                 ... 
               
               
                   
                   
                 acaaacacaaatacacacactaaattaata 
               
               
                   
               
               
                 BBa_I766556 
                 SEQ ID NO: 162 pAdh (Strong) Promoter 
                 ... 
               
               
                   
                   
                 ccaagcatacaatcaactatctcatataca 
               
               
                   
               
               
                 BBa_I766557 
                 SEQ ID NO: 163 pSte5 (Weak) Promoter 
                 ... 
               
               
                   
                   
                 gatacaggatacagcggaaacaactataa 
               
               
                   
               
               
                 BBa_J63005 
                 SEQ ID NO: 164 yeast ADH1 promoter 
                 ... 
               
               
                   
                   
                 tttcaagctataccaagcatacaatcaact 
               
               
                   
               
               
                 BBa_K105027 
                 SEQ ID NO: 165 cyc100 minimal promoter 
                 ...cctttgcagcataaattactatacttctat 
               
               
                   
               
               
                 BBa_K105028 
                 SEQ ID NO: 166 cyc70 minimal promoter 
                 ...cctttgcagcataaattactatacttctat 
               
               
                   
               
               
                 BBa_K105029 
                 SEQ ID NO: 167 cyc43 minimal promoter 
                 ...cctttgcagcataaattactatacttctat 
               
               
                   
               
               
                 BBa_K105030 
                 SEQ ID NO: 168 cyc28 minimal promoter 
                 ...cctttgcagcataaattactatacttctat 
               
               
                   
               
               
                 BBa_K105031 
                 SEQ ID NO: 169 cyc16 minimal promoter 
                 ...cctttgcagcataaattactatacttctat 
               
               
                   
               
               
                 BBa_K122000 
                 SEQ ID NO: 170 pPGK1 
                 ...ttatctactattacaacaaatataaaaca 
               
               
                   
               
               
                 BBa_K124000 
                 SEQ ID NO: 171 pCYC Yeast Promoter 
                 ... 
               
               
                   
                   
                 acaaacacaaatacacacactaaattaata 
               
               
                   
               
               
                 BBa_K124002 
                 SEQ ID NO: 172 Yeast GPD (TDH3) 
                 ... 
               
               
                   
                 Promoter 
                 gtttcgaataaacacacataaacaaacaaa 
               
               
                   
               
               
                 BBa_M31201 
                 SEQ ID NO: 173 Yeast CLB1 promoter 
                 ... 
               
               
                   
                 region, G2/M cell cycle specific 
                 accatcaaaggaagattaatcttctcata 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Examples of Constitutive Promoters from Miscellaneous Eukaryotes 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I712004 
                 SEQ ID NO: 174 CMV promoter 
                 ...agaacccactgcttactggcttatcgaaat 
               
               
                   
               
               
                 BBa_K076017 
                 SEQ ID NO: 175 Ubc Promoter 
                 ...ggccgtttaggcttttttgttagacgaag 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Examples of Cell Signaling Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I1051 
                 SEQ ID NO: 176 Lux cassette right 
                 ... 
               
               
                   
                 promoter 
                 tgttatagtcgaatacctctggcggtgata 
               
               
                   
               
               
                 BBa_I14015 
                 SEQ ID NO: 177 P(Las) TetO 
                 ... 
               
               
                   
                   
                 ttttggtacactccctatcagtgatagaga 
               
               
                   
               
               
                 BBa_I14016 
                 SEQ ID NO: 178 P(Las) CIO 
                 ... 
               
               
                   
                   
                 ctttttggtacactacctctggcggtgata 
               
               
                   
               
               
                 BBa_I14017 
                 SEQ ID NO: 179 P(Rhl) 
                 ... 
               
               
                   
                   
                 tacgcaagaaaatggtttgttatagtcgaa 
               
               
                   
               
               
                 BBa_I739105 
                 SEQ ID NO: 180 Double Promoter 
                 ... 
               
               
                   
                 (LuxR/HSL, positive/cI, negative) 
                 cgtgcgtgttgataacaccgtgcgtgttga 
               
               
                   
               
               
                 BBa_I746104 
                 SEQ ID NO: 181 P2 promoter in agr operon 
                 ... 
               
               
                   
                 from S. aureus 
                 agattgtactaaatcgtataatgacagtga 
               
               
                   
               
               
                 BBa_I751501 
                 SEQ ID NO: 182 plux-cI hybrid promoter 
                 ... 
               
               
                   
                   
                 gtgttgatgcttttatcaccgccagtggta 
               
               
                   
               
               
                 BBa_I751502 
                 SEQ ID NO: 183 plux-lac hybrid promoter 
                 ... 
               
               
                   
                   
                 agtgtgtggaattgtgagcggataacaatt 
               
               
                   
               
               
                 BBa_I761011 
                 SEQ ID NO: 184 CinR, CinL and glucose 
                 ...acatcttaaaagttttagtatcatattcgt 
               
               
                   
                 controlled promoter 
               
               
                   
               
               
                 BBa_J06403 
                 SEQ ID NO: 185 RhIR promoter repressible 
                 ... 
               
               
                   
                 by CI 
                 tacgcaagaaaatggtttgttatagtcgaa 
               
               
                   
               
               
                 BBa_J64000 
                 SEQ ID NO: 186 rhlI promoter 
                 ...atcctcctttagtcttccccctcatgtgtg 
               
               
                   
               
               
                 BBa_J64010 
                 SEQ ID NO: 187 lasI promoter 
                 ...taaaattatgaaatttgcataaattcttca 
               
               
                   
               
               
                 BBa_J64067 
                 SEQ ID NO: 188 LuxR + 3OC6HSL 
                 ...gtgttgactattttacctctggcggtgata 
               
               
                   
                 independent R0065 
               
               
                   
               
               
                 BBa_J64712 
                 SEQ ID NO: 189 LasR/LasI Inducible &amp; 
                 ... 
               
               
                   
                 RHLR/RHLI repressible Promoter 
                 gaaatctggcagtttttggtacacgaaagc 
               
               
                   
               
               
                 BBa_K091107 
                 SEQ ID NO: 190 pLux/cI Hybrid Promoter 
                 ... 
               
               
                   
                   
                 acaccgtgcgtgttgatatagtcgaataaa 
               
               
                   
               
               
                 BBa_K091117 
                 SEQ ID NO: 191 pLas promoter 
                 ...aaaattatgaaatttgtataaattcttcag 
               
               
                   
               
               
                 BBa_K091143 
                 SEQ ID NO: 192 pLas/cI Hybrid Promoter 
                 ... 
               
               
                   
                   
                 ggttattttggtacctctggcggtgataa 
               
               
                   
               
               
                 BBa_K091146 
                 SEQ ID NO: 193 pLas/Lux Hybrid Promoter 
                 ... 
               
               
                   
                   
                 tgtaggatcgtacaggtataaattcttcag 
               
               
                   
               
               
                 BBa_K091156 
                 SEQ ID NO: 194 pLux 
                 ... 
               
               
                   
                   
                 caagaaaatggtttgttatagtcgaataaa 
               
               
                   
               
               
                 BBa_K091157 
                 SEQ ID NO: 195 pLux/Las Hybrid Promoter 
                 ...ctatctcatttgctagtatagtcgaataaa 
               
               
                   
               
               
                 BBa_K145150 
                 SEQ ID NO: 196 Hybrid promoter: HSL- 
                 ...tagtttataatttaagtgttctttaatttc 
               
               
                   
                 LuxR activated, P22 C2 repressed 
               
               
                   
               
               
                 BBa_K266000 
                 SEQ ID NO: 197 PAI + LasR -&gt; LuxI (AI) 
                 ... 
               
               
                   
                   
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K266005 
                 SEQ ID NO: 198 PAI + LasR -&gt; LasI &amp; 
                 ... 
               
               
                   
                 AI + LuxR --| LasI 
                 aataactctgatagtgctagtgtagatctc 
               
               
                   
               
               
                 BBa_K266006 
                 SEQ ID NO: 199 PAI + LasR -&gt; LasI + GFP &amp; 
                 ... 
               
               
                   
                 AI + LuxR --| LasI + GFP 
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K266007 
                 SEQ ID NO: 200 Complex QS -&gt; LuxI &amp; 
                 ... 
               
               
                   
                 LasI circuit 
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_R0061 
                 SEQ ID NO: 201 Promoter (HSL-mediated 
                 ttgacacctgtaggatcgtacaggtataat 
               
               
                   
                 luxR repressor) 
               
               
                   
               
               
                 BBa_R0062 
                 SEQ ID NO: 202 Promoter (luxR &amp; HSL 
                 ... 
               
               
                   
                 regulated -- lux pR) 
                 caagaaaatggtttgttatagtcgaataaa 
               
               
                   
               
               
                 BBa_R0063 
                 SEQ ID NO: 203 Promoter (luxR &amp; HSL 
                 ... 
               
               
                   
                 regulated -- lux pL) 
                 cacgcaaaacttgcgacaaacaataggtaa 
               
               
                   
               
               
                 BBa_R0071 
                 SEQ ID NO: 204 Promoter (RhlR &amp; C4- 
                 ... 
               
               
                   
                 HSL regulated) 
                 gttagctttcgaattggctaaaaagtgttc 
               
               
                   
               
               
                 BBa_R0078 
                 SEQ ID NO: 205 Promoter (cinR and HSL 
                 ... 
               
               
                   
                 regulated) 
                 ccattctgctttccacgaacttgaaaacgc 
               
               
                   
               
               
                 BBa_R0079 
                 SEQ ID NO: 206 Promoter (LasR &amp; PAI 
                 ... 
               
               
                   
                 regulated) 
                 ggccgcgggttattttggtacacgaaagc 
               
               
                   
               
               
                 BBa_R1062 
                 SEQ ID NO: 207 Promoter, Standard (luxR 
                 ... 
               
               
                   
                 and HSL regulated -- lux pR) 
                 aagaaaatggtttgttgatactcgaataaa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Examples of Metal Inducible Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I721001 
                 SEQ ID NO: 208 Lead Promoter 
                 ...gaaaaccttgtcaatgaagagcgatctatg 
               
               
                   
               
               
                 BBa_I731004 
                 SEQ ID NO: 209 FecA promoter 
                 ...ttctcgttcgactcatagctgaacacaaca 
               
               
                   
               
               
                 BBa_I760005 
                 SEQ ID NO: 210 Cu-sensitive promoter 
                 atgacaaaattgtcat 
               
               
                   
               
               
                 BBa_I765000 
                 SEQ ID NO: 211 Fe promoter 
                 ... 
               
               
                   
                   
                 accaatgctgggaacggccagggcacctaa 
               
               
                   
               
               
                 BBa_I765007 
                 SEQ ID NO: 212 Fe and UV promoters 
                 ...ctgaaagcgcataccgctatggagggggtt 
               
               
                   
               
               
                 BBa_J3902 
                 SEQ ID NO: 213 PrFe (PI + PII rus 
                 ...tagatatgcctgaaagcgcataccgctatg 
               
               
                   
                 operon) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Examples of T7 Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I712074 
                 SEQ ID NO: 214 T7 promoter (strong 
                 ...agggaatacaagctacttgttctattgca 
               
               
                   
                 promoter from T7 bacteriophage) 
               
               
                   
               
               
                 BBa_I719005 
                 SEQ ID NO: 215 T7 Promoter 
                 taatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_J34814 
                 SEQ ID NO: 216 T7 Promoter 
                 gaatttaatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_J64997 
                 SEQ ID NO: 217 T7 consensus-10 and rest 
                 taatacgactcactatagg 
               
               
                   
               
               
                 BBa_J64998 
                 SEQ ID NO: 218 consensus-10 and rest 
                 atttaggtgacactataga 
               
               
                   
                 from SP6 
               
               
                   
               
               
                 BBa_K113010 
                 SEQ ID NO: 219 overlapping T7 promoter 
                 ... 
               
               
                   
                   
                 gagtcgtattaatacgactcactatagggg 
               
               
                   
               
               
                 BBa_K113011 
                 SEQ ID NO: 220 more overlapping T7 
                 ... 
               
               
                   
                 promoter 
                 agtgagtcgtactacgactcactatagggg 
               
               
                   
               
               
                 BBa_K113012 
                 SEQ ID NO: 221 weaken overlapping T7 
                 ... 
               
               
                   
                 promoter 
                 gagtcgtattaatacgactctctatagggg 
               
               
                   
               
               
                 BBa_R0085 
                 SEQ ID NO: 222 T7 Consensus Promoter 
                 taatacgactcactatagggaga 
               
               
                   
                 Sequence 
               
               
                   
               
               
                 BBa_R0180 
                 SEQ ID NO: 223 T7 RNAP promoter 
                 ttatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_R0181 
                 SEQ ID NO: 224 T7 RNAP promoter 
                 gaatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_R0182 
                 SEQ ID NO: 225 T7 RNAP promoter 
                 taatacgtctcactatagggaga 
               
               
                   
               
               
                 BBa_R0183 
                 SEQ ID NO: 226 T7 RNAP promoter 
                 tcatacgactcactatagggaga 
               
               
                   
               
               
                 BBa_R0184 
                 SEQ ID NO: 227 T7 promoter (ladI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_R0185 
                 SEQ ID NO: 228 T7 promoter (ladI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_R0186 
                 SEQ ID NO: 229 T7 promoter (ladI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_R0187 
                 SEQ ID NO: 230 T7 promoter (ladI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_Z0251 
                 SEQ ID NO: 231 T7 strong promoter 
                 ... 
               
               
                   
                   
                 taatacgactcactatagggagaccacaac 
               
               
                   
               
               
                 BBa_Z0252 
                 SEQ ID NO: 232 T7 weak binding and 
                 ... 
               
               
                   
                 processivity 
                 taattgaactcactaaagggagaccacagc 
               
               
                   
               
               
                 BBa_Z0253 
                 SEQ ID NO: 233 T7 weak binding promoter 
                 ... 
               
               
                   
                   
                 cgaagtaatacgactcactattagggaaga 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Examples of Stress Kit Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K086017 
                 SEQ ID NO: 234 unmodified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter 
                 ttgtgagcggataacaagatactgagcaca 
               
               
                   
               
               
                 BBa_K086018 
                 SEQ ID NO: 235 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ24 
                 ttgtgagcggataacaattctgaagaacaa 
               
               
                   
               
               
                 BBa_K086019 
                 SEQ ID NO: 236 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ24 
                 ttgtgagcggataacaattctgataaaaca 
               
               
                   
               
               
                 BBa_K086020 
                 SEQ ID NO: 237 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ24 
                 ttgtgagcggataacatctaaccattaga 
               
               
                   
               
               
                 BBa_K086021 
                 SEQ ID NO: 238 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ24 
                 ttgtgagcggataacatagcagataagaaa 
               
               
                   
               
               
                 BBa_K086022 
                 SEQ ID NO: 239 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ28 
                 gtttgagcgagtaacgccgaaaatcttgca 
               
               
                   
               
               
                 BBa_K086023 
                 SEQ ID NO: 240 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ28 
                 gtgtgagcgagtaacgacgaaaatcttgca 
               
               
                   
               
               
                 BBa_K086024 
                 SEQ ID NO: 241 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ28 
                 tttgagcgagtaacagccgaaaatcttgca 
               
               
                   
               
               
                 BBa_K086025 
                 SEQ ID NO: 242 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ28 
                 tgtgagcgagtaacagccgaaaatcttgca 
               
               
                   
               
               
                 BBa_K086026 
                 SEQ ID NO: 243 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtggcaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086027 
                 SEQ ID NO: 244 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtgacaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086028 
                 SEQ ID NO: 245 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtaacaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086029 
                 SEQ ID NO: 246 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtaacaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086030 
                 SEQ ID NO: 247 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ38 
                 cagtgagcgagtaacaactacgctgtttta 
               
               
                   
               
               
                 BBa_K086031 
                 SEQ ID NO: 248 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ38 
                 cagtgagcgagtaacaactacgctgtttta 
               
               
                   
               
               
                 BBa_K086032 
                 SEQ ID NO: 249 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ38 
                 atgtgagcggataacactataattaataga 
               
               
                   
               
               
                 BBa_K086033 
                 SEQ ID NO: 250 modified Lutz-Bujard 
                 ... 
               
               
                   
                 LacO promoter, with alternative sigma factor σ38 
                 atgtgagcggataacactataattaataga 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Examples of Logic Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I732200 
                 SEQ ID NO: 251 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O1wt1) 
                 gaattgtgagcggataacaattggatccgg 
               
               
                   
               
               
                 BBa_I732201 
                 SEQ ID NO: 252 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O11) 
                 ggaattgtgagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732202 
                 SEQ ID NO: 253 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O22) 
                 ggaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732203 
                 SEQ ID NO: 254 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O33) 
                 ggaattgtaaacgtttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732204 
                 SEQ ID NO: 255 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O44) 
                 ggaattgtgaacgttcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732205 
                 SEQ ID NO: 256 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O55) 
                 ggaattttgagcgctcaaaattggatccgg 
               
               
                   
               
               
                 BBa_I732206 
                 SEQ ID NO: 257 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O66) 
                 ggaattatgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732207 
                 SEQ ID NO: 258 NOT Gate Promoter 
                 ... 
               
               
                   
                 Family Member (D001O77) 
                 gggacgactgtatacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732270 
                 SEQ ID NO: 259 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O12) 
                 ggaattgtgagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732271 
                 SEQ ID NO: 260 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O16) 
                 ggaattgtgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732272 
                 SEQ ID NO: 261 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O17) 
                 ggaattgtgagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732273 
                 SEQ ID NO: 262 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O21) 
                 ggaattgtaagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732274 
                 SEQ ID NO: 263 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O24) 
                 ggaattgtaagcgttcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732275 
                 SEQ ID NO: 264 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O26) 
                 ggaattgtaagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732276 
                 SEQ ID NO: 265 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O27) 
                 ggaattgtaagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732277 
                 SEQ ID NO: 266 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O46) 
                 ggaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732278 
                 SEQ ID NO: 267 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O47) 
                 ggaattgtgaactacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732279 
                 SEQ ID NO: 268 Promoter Family Member 
                 ... 
               
               
                   
                 with Hybrid Operator (D001O61) 
                 ggaattatgagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732301 
                 SEQ ID NO: 269 NAND Candidate 
                 ... 
               
               
                   
                 (U073O26D001O16) 
                 ggaattgtgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732302 
                 SEQ ID NO: 270 NAND Candidate 
                 ... 
               
               
                   
                 (U073O27D001O17) 
                 ggaattgtgagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732303 
                 SEQ ID NO: 271 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D001O46) 
                 ggaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732304 
                 SEQ ID NO: 272 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D001O47) 
                 ggaattgtgaactacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732305 
                 SEQ ID NO: 273 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D059O46) 
                 taaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732306 
                 SEQ ID NO: 274 NAND Candidate 
                 ... 
               
               
                   
                 (U073O11D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732351 
                 SEQ ID NO: 275 NOR Candidate 
                 ... 
               
               
                   
                 (U037O11D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732352 
                 SEQ ID NO: 276 NOR Candidate 
                 ... 
               
               
                   
                 (U035O44D001O22) 
                 ggaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732400 
                 SEQ ID NO: 277 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732401 
                 SEQ ID NO: 278 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732402 
                 SEQ ID NO: 279 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732403 
                 SEQ ID NO: 280 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732404 
                 SEQ ID NO: 281 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732405 
                 SEQ ID NO: 282 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732406 
                 SEQ ID NO: 283 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732407 
                 SEQ ID NO: 284 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732408 
                 SEQ ID NO: 285 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732409 
                 SEQ ID NO: 286 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732410 
                 SEQ ID NO: 287 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732411 
                 SEQ ID NO: 288 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732412 
                 SEQ ID NO: 289 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732413 
                 SEQ ID NO: 290 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732414 
                 SEQ ID NO: 291 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732415 
                 SEQ ID NO: 292 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732416 
                 SEQ ID NO: 293 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732417 
                 SEQ ID NO: 294 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732418 
                 SEQ ID NO: 295 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732419 
                 SEQ ID NO: 296 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732420 
                 SEQ ID NO: 297 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732421 
                 SEQ ID NO: 298 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732422 
                 SEQ ID NO: 299 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732423 
                 SEQ ID NO: 300 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732424 
                 SEQ ID NO: 301 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732425 
                 SEQ ID NO: 302 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732426 
                 SEQ ID NO: 303 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732427 
                 SEQ ID NO: 304 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732428 
                 SEQ ID NO: 305 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732429 
                 SEQ ID NO: 306 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732430 
                 SEQ ID NO: 307 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732431 
                 SEQ ID NO: 308 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732432 
                 SEQ ID NO: 309 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732433 
                 SEQ ID NO: 310 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732434 
                 SEQ ID NO: 311 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732435 
                 SEQ ID NO: 312 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732436 
                 SEQ ID NO: 313 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732437 
                 SEQ ID NO: 314 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732438 
                 SEQ ID NO: 315 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732439 
                 SEQ ID NO: 316 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732440 
                 SEQ ID NO: 317 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732441 
                 SEQ ID NO: 318 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732442 
                 SEQ ID NO: 319 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732443 
                 SEQ ID NO: 320 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732444 
                 SEQ ID NO: 321 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732445 
                 SEQ ID NO: 322 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732446 
                 SEQ ID NO: 323 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732447 
                 SEQ ID NO: 324 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732448 
                 SEQ ID NO: 325 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732450 
                 SEQ ID NO: 326 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O26 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732451 
                 SEQ ID NO: 327 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O27 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732452 
                 SEQ ID NO: 328 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O26 + D062O61) 
                 caaattatgagcgctcacaattggatccgg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Examples of Positively Regulated  E. coli  σ70 Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I0500 
                 SEQ ID NO: 329 Inducible pBad/araC 
                 ...gatctccatacccgtttttttgggctagc 
               
               
                   
                 promoter 
               
               
                   
               
               
                 BBa_I1051 
                 SEQ ID NO: 330 Lux cassette right 
                 ...tgttatagtcgaatacctctggcggtgata 
               
               
                   
                 promoter 
               
               
                   
               
               
                 BBa_I12006 
                 SEQ ID NO: 331 Modified lamdba Prm 
                 ...attacaaactttcttgtatagatttaacgt 
               
               
                   
                 promoter (repressed by 434 cI) 
               
               
                   
               
               
                 BBa_I12007 
                 SEQ ID NO: 332 Modified lambda Prm 
                 ...atttataaatagtggtgatagatttaacgt 
               
               
                   
                 promoter (OR-3 obliterated) 
               
               
                   
               
               
                 BBa_I12036 
                 SEQ ID NO: 333 Modified lamdba Prm 
                 ...tttcttgtatagatttacaatgtatcttgt 
               
               
                   
                 promoter (cooperative repression by 434 cI) 
               
               
                   
               
               
                 BBa_I12040 
                 SEQ ID NO: 334 Modified lambda 
                 ...tttcttgtagatacttacaatgtatcttgt 
               
               
                   
                 P(RM) promoter: −10 region from P(L) and 
               
               
                   
                 cooperatively repressed by 434 cI 
               
               
                   
               
               
                 BBa_I12210 
                 SEQ ID NO: 335 plac Or2-62 (positive) 
                 ...ctttatgcttccggctcgtatgttgtgtgg 
               
               
                   
               
               
                 BBa_I13406 
                 SEQ ID NO: 336 Pbad/AraC with extra 
                 ...ttttttgggctagcaagatttaccatggat 
               
               
                   
                 REN sites 
               
               
                   
               
               
                 BBa_I13453 
                 SEQ ID NO: 337 Pbad promoter 
                 ...tgtttctccataccgtttttttgggctagc 
               
               
                   
               
               
                 BBa_I14015 
                 SEQ ID NO: 338 P(Las) TetO 
                 ...ttttggtacactccctatcagtgatagaga 
               
               
                   
               
               
                 BBa_I14016 
                 SEQ ID NO: 339 P(Las) CIO 
                 ...ctttttggtacactacctctggcggtgata 
               
               
                   
               
               
                 BBa_I14017 
                 SEQ ID NO: 340 P(Rhl) 
                 ...tacgcaagaaaatggtttgttatagtcgaa 
               
               
                   
               
               
                 BBa_I721001 
                 SEQ ID NO: 341 Lead Promoter 
                 ...gaaaaccttgtcaatgaagagcgatctatg 
               
               
                   
               
               
                 BBa_I723020 
                 SEQ ID NO: 342 Pu 
                 ...ctcaaagcgggccagccgtagccgttacgc 
               
               
                   
               
               
                 BBa_I731004 
                 SEQ ID NO: 343 FecA promoter 
                 ...ttctcgttcgactcatagctgaacacaaca 
               
               
                   
               
               
                 BBa_I1739104 
                 SEQ ID NO: 344 Double Promoter 
                 ...gttctttaattatttaagtgttctttaatt 
               
               
                   
                 (LuxR/HSL, positive/P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739105 
                 SEQ ID NO: 345 Double Promoter 
                 ...cgtgcgtgttgataacaccgtgcgtgttga 
               
               
                   
                 (LuxR/HSL, positive/cI, negative) 
               
               
                   
               
               
                 BBa_I741018 
                 SEQ ID NO: 346 Right facing promoter 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 (for xylF) controlled by xylR and CRP-cAMP 
               
               
                   
               
               
                 BBa_I741019 
                 SEQ ID NO: 347 Right facing promoter 
                 ...gcaaaataaaatggaatgatgaaactgggt 
               
               
                   
                 (for xylA) controlled by xylR and CRP-cAMP 
               
               
                   
               
               
                 BBa_I741020 
                 SEQ ID NO: 348 promoter to xylF 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 without CRP and several binding sites for xylR 
               
               
                   
               
               
                 BBa_I741021 
                 SEQ ID NO: 349 promoter to xylA 
                 ...atttcacactgctattgagataattcacaa 
               
               
                   
                 without CRP and several binding sites for xylR 
               
               
                   
               
               
                 BBa_I746104 
                 SEQ ID NO: 350 P2 promoter in agr 
                 ...agattgtactaaatcgtataatgacagtga 
               
               
                   
                 operon from  S. aureus   
               
               
                   
               
               
                 BBa_I746360 
                 SEQ ID NO: 351 PF promoter from P2 
                 ...gacatctccggcgcaactgaaaataccact 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_I746361 
                 SEQ ID NO: 352 PO promoter from P2 
                 ...gaggatgcgcatcgtcgggaaactgatgcc 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_I746362 
                 SEQ ID NO: 353 PP promoter from P2 
                 ...catccgggactgatggcggaggatgcgcat 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_I746363 
                 SEQ ID NO: 354 PV promoter from P2 
                 ...aacttttatatattgtgcaatctcacatgc 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_I746364 
                 SEQ ID NO: 355 Psid promoter from P4 
                 ...tgttgtccggtgtacgtcacaattttctta 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_I746365 
                 SEQ ID NO: 356 PLL promoter from P4 
                 ...aatggctgtgtgttttttgttcatctccac 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_I751501 
                 SEQ ID NO: 357 plux-cI hybrid 
                 ...gtgttgatgcttttatcaccgccagtggta 
               
               
                   
                 promoter 
               
               
                   
               
               
                 BBa_I751502 
                 SEQ ID NO: 358 plux-lac hybrid 
                 ...agtgtgtggaattgtgagcggataacaatt 
               
               
                   
                 promoter 
               
               
                   
               
               
                 BBa_I760005 
                 SEQ ID NO: 359 Cu-sensitive promoter 
                 atgacaaaattgtcat 
               
               
                   
               
               
                 BBa_I761011 
                 SEQ ID NO: 360 CinR, CinL and 
                 ...acatcttaaaagttttagtatcatattcgt 
               
               
                   
                 glucose controlled promoter 
               
               
                   
               
               
                 BBa_I765001 
                 SEQ ID NO: 361 UV promoter 
                 ...ctgaaagcgcataccgctatggagggggtt 
               
               
                   
               
               
                 BBa_I765007 
                 SEQ ID NO: 362 Fe and UV promoters 
                 ...ctgaaagcgcataccgctatggagggggtt 
               
               
                   
               
               
                 BBa_J01005 
                 SEQ ID NO: 363 pspoIIE promoter 
                 ...aacgaatataacaggtgggagatgagagga 
               
               
                   
                 (spo0A J01004, positive) 
               
               
                   
               
               
                 BBa_J03007 
                 SEQ ID NO: 364 Maltose specific 
                 ...aatatttcctcattttccacagtgaagtga 
               
               
                   
                 promoter 
               
               
                   
               
               
                 BBa_J06403 
                 SEQ ID NO: 365 RhIR promoter 
                 ...tacgcaagaaaatggtttgttatagtcgaa 
               
               
                   
                 repressible by CI 
               
               
                   
               
               
                 BBa_J07007 
                 SEQ ID NO: 366 ctx promoter 
                 ...atttaattgttttgatcaattatttttctg 
               
               
                   
               
               
                 BBa_J13210 
                 SEQ ID NO: 367 pOmpR dependent 
                 ...attattctgcatttttggggagaatggact 
               
               
                   
                 POPS producer 
               
               
                   
               
               
                 BBa_J15502 
                 SEQ ID NO: 368 copA promoter 
                 ...ccttgctggaaggtttaacctttatcacag 
               
               
                   
               
               
                 BBa_J16101 
                 SEQ ID NO: 369 BanAp - Banana- 
                 atgatgtgtccatggatta 
               
               
                   
                 induced Promoter 
               
               
                   
               
               
                 BBa_J16105 
                 SEQ ID NO: 370 HelPp - “Help” 
                 atgatagacgatgtgcggacaacgtg 
               
               
                   
                 Dependant promoter 
               
               
                   
               
               
                 BBa_J45503 
                 SEQ ID NO: 371 hybB Cold Shock 
                 ...cattagccgccaccatggggttaagtagca 
               
               
                   
                 Promoter 
               
               
                   
               
               
                 BBa_J58100 
                 SEQ ID NO: 372 AND-type promoter 
                 ...atttataaatagtggtgatagatttaacgt 
               
               
                   
                 synergistically activated by cI and CRP 
               
               
                   
               
               
                 BBa_J61051 
                 SEQ ID NO: 373 [Psal1] 
                 ...ataaagccatcacgagtaccatagaggatc 
               
               
                   
               
               
                 BBa_J61054 
                 SEQ ID NO: 374 [HIP-1] Promoter 
                 ...tttgtcttttcttgcttaataatgttgtca 
               
               
                   
               
               
                 BBa_J61055 
                 SEQ ID NO: 375 [HIP-1 fnr] Promoter 
                 ...tttgtcttttcttgcttaataatgttgtca 
               
               
                   
               
               
                 BBa_J64000 
                 SEQ ID NO: 376 rhlI promoter 
                 ...atcctcctttagtcttccccctcatgtgtg 
               
               
                   
               
               
                 BBa_J64010 
                 SEQ ID NO: 377 lasI promoter 
                 ...taaaattatgaaatttgcataaattcttca 
               
               
                   
               
               
                 BBa_J64712 
                 SEQ ID NO: 378 LasR/LasI Inducible &amp; 
                 ...gaaatctggcagtttttggtacacgaaagc 
               
               
                   
                 RHLR/RHLI repressible Promoter 
               
               
                   
               
               
                 BBa_J64800 
                 SEQ ID NO: 379 RHLR/RHLI Inducible 
                 ...tgccagttctggcaggtctaaaaagtgttc 
               
               
                   
                 &amp; LasR/LasI repressible Promoter 
               
               
                   
               
               
                 BBa_J64804 
                 SEQ ID NO: 380 The promoter region 
                 ...cacagaacttgcatttatataaagggaaag 
               
               
                   
                 (inclusive of regulator binding sites) 
               
               
                   
                 of the  B. subtilis  RocDEF operon 
               
               
                   
               
               
                 BBa_K091107 
                 SEQ ID NO: 381 pLux/cI Hybrid 
                 ...acaccgtgcgtgttgatatagtcgaataaa 
               
               
                   
                 Promoter 
               
               
                   
               
               
                 BBa_K091117 
                 SEQ ID NO: 382 pLas promoter 
                 ...aaaattatgaaatttgtataaattcttcag 
               
               
                   
               
               
                 BBa_K091143 
                 SEQ ID NO: 383 pLas/cI Hybrid 
                 ...ggttctttttggtacctctggcggtgataa 
               
               
                   
                 Promoter 
               
               
                   
               
               
                 BBa_K091146 
                 SEQ ID NO: 384 pLas/Lux Hybrid 
                 ...tgtaggatcgtacaggtataaattcttcag 
               
               
                   
                 Promoter 
               
               
                   
               
               
                 BBa_K091156 
                 SEQ ID NO: 385 pLux 
                 ...caagaaaatggtttgttatagtcgaataaa 
               
               
                   
               
               
                 BBa_K091157 
                 SEQ ID NO: 386 pLux/Las Hybrid 
                 ...ctatctcatttgctagtatagtcgaataaa 
               
               
                   
                 Promoter 
               
               
                   
               
               
                 BBa_K100000 
                 SEQ ID NO: 387 Natural Xylose 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 Regulated Bi-Directional Operator 
               
               
                   
               
               
                 BBa_K100001 
                 SEQ ID NO: 388 Edited Xylose 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 Regulated Bi-Directional Operator 1 
               
               
                   
               
               
                 BBa_K100002 
                 SEQ ID NO: 389 Edited Xylose 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 Regulated Bi-Directional Operator 2 
               
               
                   
               
               
                 BBa_K112118 
                 SEQ ID NO: 390 rrnB P1 promoter 
                 ...ataaatgcttgactctgtagcgggaaggcg 
               
               
                   
               
               
                 BBa_K112320 
                 SEQ ID NO: 391 {&lt;ftsAZ promoter&gt;} 
                 ...aaaactggtagtaggactggagattggtac 
               
               
                   
                 in BBb format 
               
               
                   
               
               
                 BBa_K112322 
                 SEQ ID NO: 392 {Pdps} in BBb format 
                 ...gggacacaaacatcaagaggatatgagatt 
               
               
                   
               
               
                 BBa_K112402 
                 SEQ ID NO: 393 promoter for FabA 
                 ...gtcaaaatgaccgaaacgggtggtaacttc 
               
               
                   
                 gene - Membrane Damage and Ultrasound 
               
               
                   
                 Sensitive 
               
               
                   
               
               
                 BBa_K112405 
                 SEQ ID NO: 394 Promoter for CadA and 
                 ...agtaatcttatcgccagtttggtctggtca 
               
               
                   
                 CadB genes 
               
               
                   
               
               
                 BBa_K112406 
                 SEQ ID NO: 395 cadC promoter 
                 ...agtaatcttatcgccagtttggtctggtca 
               
               
                   
               
               
                 BBa_K112701 
                 SEQ ID NO: 396 hns promoter 
                 ...aattctgaacaacatccgtactcttcgtgc 
               
               
                   
               
               
                 BBa_K112900 
                 SEQ ID NO: 397 Pbad 
                 ...tcgataagattaccgatcttacctgaagct 
               
               
                   
               
               
                 BBa_K116001 
                 SEQ ID NO: 398 nhaA promoter, which 
                 ...cgatctattcacctgaaagagaaataaaaa 
               
               
                   
                 can be regulated by pH and nhaR protein. 
               
               
                   
               
               
                 BBa_K116401 
                 SEQ ID NO: 399 external phosphate 
                 ...atcgcaacctatttattacaacactagtgc 
               
               
                   
                 sensing promoter 
               
               
                   
               
               
                 BBa_K116500 
                 SEQ ID NO: 400 OmpF promoter that is 
                 ...aaacgttagtttgaatggaaagatgcctgc 
               
               
                   
                 activated or repressed by OmpR according to 
               
               
                   
                 osmolarity. 
               
               
                   
               
               
                 BBa_K116603 
                 SEQ ID NO: 401 pRE promoter from λ 
                 ...tttgcacgaaccatatgtaagtatttcctt 
               
               
                   
                 phage 
               
               
                   
               
               
                 BBa_K117002 
                 SEQ ID NO: 402 LsrA promoter 
                 ...taacacttatttaattaaaaagaggagaaa 
               
               
                   
                 (indirectly activated by AI-2) 
               
               
                   
               
               
                 BBa_K118011 
                 SEQ ID NO: 403 PcstA (glucose- 
                 ...tagaaacaaaatgtaacatctctatggaca 
               
               
                   
                 repressible promoter) 
               
               
                   
               
               
                 BBa_K121011 
                 SEQ ID NO: 404 promoter (ladI 
                 ...acaggaaacagctatgaccatgattacgcc 
               
               
                   
                 regulated) 
               
               
                   
               
               
                 BBa_K135000 
                 SEQ ID NO: 405 pCpxR (CpxR 
                 ...agcgacgtctgatgacgtaatttctgcctc 
               
               
                   
                 responsive promoter) 
               
               
                   
               
               
                 BBa_K136010 
                 SEQ ID NO: 406 fliA promoter 
                 ...gttcactctataccgctgaaggtgtaatgg 
               
               
                   
               
               
                 BBa_K145150 
                 SEQ ID NO: 407 Hybrid promoter: 
                 ...tagtttataatttaagtgttctttaatttc 
               
               
                   
                 HSL-LuxR activated, P22 C2 repressed 
               
               
                   
               
               
                 BBa_K180000 
                 SEQ ID NO: 408 Hybrid promoter (trp 
                 ...cgagcacttcaccaacaaggaccatagcat 
               
               
                   
                 &amp; lac regulated -- tac pR) 
               
               
                   
               
               
                 BBa_K180002 
                 SEQ ID NO: 409 tac pR testing plasmid 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 (GFP) 
               
               
                   
               
               
                 BBa_K180003 
                 SEQ ID NO: 410 PTAC testing plasmid 
                 ...catggcatggatgaactatacaaataataa 
               
               
                   
                 (GFP) - basic 
               
               
                   
               
               
                 BBa_K180004 
                 SEQ ID NO: 411 Game of Life - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Primary plasmid 
               
               
                   
               
               
                 BBa_K180005 
                 SEQ ID NO: 412 GoL - Primary plasmid 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 (part 1)/RPS - Paper primary plasmid (part 1) 
               
               
                   
                 [LuxR generator] 
               
               
                   
               
               
                 BBa_K180006 
                 SEQ ID NO: 413 Game of Life - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Primary plasmid (part 2) [lux pR, GFP and LacI 
               
               
                   
                 generator] 
               
               
                   
               
               
                 BBa_K180007 
                 SEQ ID NO: 414 Game of Life - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Secondary plasmid [tac pR, LuxI generator] 
               
               
                   
               
               
                 BBa_K180010 
                 SEQ ID NO: 415 Rock-paper-scissors - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Rock primary plasmid 
               
               
                   
               
               
                 BBa_K180011 
                 SEQ ID NO: 416 Rock - Primary 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 plasmid (part 1) [RhlR generator] 
               
               
                   
               
               
                 BBa_K180012 
                 SEQ ID NO: 417 Rock - Primary 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 plasmid (part 2) [tac pR, mCherry and LasI 
               
               
                   
                 generator] 
               
               
                   
               
               
                 BBa_K180013 
                 SEQ ID NO: 418 Rock-paper-scissors - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Rock secondary plasmid [rhl pR, LacI generator] 
               
               
                   
               
               
                 BBa_K180014 
                 SEQ ID NO: 419 Rock-paper-scissors - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Paper primary plasmid 
               
               
                   
               
               
                 BBa_K180015 
                 SEQ ID NO: 420 Paper - Primary 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 plasmid (part 2) [tac pR, GFP and RhlI generator] 
               
               
                   
               
               
                 BBa_K180016 
                 SEQ ID NO: 421 Rock-paper-scissors - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Paper secondary plasmid [lux pR, LacI generator] 
               
               
                   
               
               
                 BBa_K180017 
                 SEQ ID NO: 422 Rock-paper-scissors - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Scissors primary plasmid 
               
               
                   
               
               
                 BBa_K180018 
                 SEQ ID NO: 423 Scissors - Primary 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 plasmid (part 1) [LasR generator] 
               
               
                   
               
               
                 BBa_K180019 
                 SEQ ID NO: 424 Scissors - Primary 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 plasmid (part 2) [tac pR, mBanana and LuxI 
               
               
                   
                 generator] 
               
               
                   
               
               
                 BBa_K180020 
                 SEQ ID NO: 425 Rock-paper-scissors - 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 Scissors secondary plasmid [las pR, LacI 
               
               
                   
                 generator] 
               
               
                   
               
               
                 BBa_K206000 
                 SEQ ID NO: 426 pBAD strong 
                 ...tgtttctccataccgtttttttgggctagc 
               
               
                   
               
               
                 BBa_K206001 
                 SEQ ID NO: 427 pBAD weak 
                 ...tgtttctccataccgtttttttgggctagc 
               
               
                   
               
               
                 BBa_K259005 
                 SEQ ID NO: 428 AraC Rheostat 
                 ...ttttatcgcaactctctactgtttctccat 
               
               
                   
                 Promoter 
               
               
                   
               
               
                 BBa_K259007 
                 SEQ ID NO: 429 AraC Promoter fused 
                 ...gtttctccattactagagaaagaggggaca 
               
               
                   
                 with RBS 
               
               
                   
               
               
                 BBa_K266000 
                 SEQ ID NO: 430 PAI + LasR -&gt; LuxI 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 (AI) 
               
               
                   
               
               
                 BBa_K266005 
                 SEQ ID NO: 431 PAI + LasR -&gt; LasI &amp; 
                 ...aataactctgatagtgctagtgtagatctc 
               
               
                   
                 AI + LuxR--| LasI 
               
               
                   
               
               
                 BBa_K266006 
                 SEQ ID NO: 432 PAI + LasR -&gt; 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 LasI + GFP &amp; AI + LuxR--| LasI + GFP 
               
               
                   
               
               
                 BBa_K266007 
                 SEQ ID NO: 433 Complex QS -&gt; LuxI 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 &amp; LasI circuit 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Examples of Positively regulated 
               
               
                   E. coli  σS promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K112322 
                 SEQ ID NO: 434 
                 ...gggacacaaacatc 
               
               
                   
                 {Pdps} in BBb 
                 aagaggatatgagatt 
               
               
                   
                 format 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Examples of Positively regulated 
               
               
                   E. coli  σ32 promoters 
               
            
           
           
               
               
               
            
               
                   
                   
                 Promoter 
               
               
                 Name 
                 Description 
                 Sequence 
               
               
                   
               
               
                 BBa_K112400 
                 SEQ ID NO: 435 
                 ... 
               
               
                   
                 Promoter for grpE 
                 ataataagcgaagttag 
               
               
                   
                 gene - Heat Shock and 
                 cgagatgaatgcg 
               
               
                   
                 Ultrasound Sensitive 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Examples of Positively regulated 
               
               
                   E. coli  σ54 promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J64979 
                 SEQ ID NO: 
                 ...agttggcacagatttcgcttt 
               
               
                   
                 436 glnAp2 
                 atctttttt 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Examples of Positively regulated  B. subtilis  σA promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_R0062 
                 SEQ ID NO: 437 Promoter (luxR &amp; HSL regulated -- 
                 ... 
               
               
                   
                 lux pR) 
                 caagaaaatggtttgttatagtcgaataaa 
               
               
                   
               
               
                 BBa_R0065 
                 SEQ ID NO: 438 Promoter (lambda cI and luxR 
                 ...gtgttgactattttacctctggcggtgata 
               
               
                   
                 regulated -- hybrid) 
               
               
                   
               
               
                 BBa_R0071 
                 SEQ ID NO: 439 Promoter (RhlR &amp; C4-HSL 
                 ... 
               
               
                   
                 regulated) 
                 gttagctttcgaattggctaaaaagtgttc 
               
               
                   
               
               
                 BBa_R0078 
                 SEQ ID NO: 440 Promoter (cinR and HSL 
                 ... 
               
               
                   
                 regulated) 
                 ccattctgctttccacgaacttgaaaacgc 
               
               
                   
               
               
                 BBa_R0079 
                 SEQ ID NO: 441 Promoter (LasR &amp; PAI 
                 ... 
               
               
                   
                 regulated) 
                 ggccgcgggttattttggtacacgaaagc 
               
               
                   
               
               
                 BBa_R0080 
                 SEQ ID NO: 442 Promoter (AraC regulated) 
                 ...ttttatcgcaactctctactgtttctccat 
               
               
                   
               
               
                 BBa_R0082 
                 SEQ ID NO: 443 Promoter (OmpR, positive) 
                 ...attattctgcatttttggggagaatggact 
               
               
                   
               
               
                 BBa_R0083 
                 SEQ ID NO: 444 Promoter (OmpR, positive) 
                 ...attattctgcatttttggggagaatggact 
               
               
                   
               
               
                 BBa_R0084 
                 SEQ ID NO: 445 Promoter (OmpR, positive) 
                 ... 
               
               
                   
                   
                 aacgttagtttgaatggaaagatgcctgca 
               
               
                   
               
               
                 BBa_R1062 
                 SEQ ID NO: 446 Promoter, Standard (luxR and 
                 ... 
               
               
                   
                 HSL regulated -- lux pR) 
                 aagaaaatggtttgttgatactcgaataaa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Examples of Miscellaneous Prokaryotic Induced Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J64001 
                 SEQ ID NO: 447 psicA from  Salmonella   
                 ...aacgcagtcgttaagttctacaaagtcggt 
               
               
                   
               
               
                 BBa_J64750 
                 SEQ ID NO: 448 SPI-1 TTSS secretion-linked 
                 ... 
               
               
                   
                 promoter from  Salmonella   
                 gtcggtgacagataacaggagtaagtaatg 
               
               
                   
               
               
                 BBa_K112149 
                 SEQ ID NO: 449 PmgtCB Magnesium promoter 
                 ...tattggctgactataataagcgcaaattca 
               
               
                   
                 from  Salmonella   
               
               
                   
               
               
                 BBa_K116201 
                 SEQ ID NO: 450 ureD promoter from  P   
               
               
                   
                 
                   mirabilis 
                 
               
               
                   
               
               
                 BBa_K125100 
                 SEQ ID NO: 451 nir promoter 
                 ...cgaaacgggaaccctatattgatctctact 
               
               
                   
                 from  Synechocystis  sp. PCC6803 
               
               
                   
               
               
                 BBa_K131017 
                 SEQ ID NO: 452 p_qrr4 from  Vibrio harveyi   
                 ...aagttggcacgcatcgtgctttatacagat 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Examples of Yeast Positive (Activatible) Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J63006 
                 SEQ ID NO: 453 yeast GAL1 promoter 
                 . . . gaggaaactagacccgccgccaccatggag 
               
               
                   
               
               
                 BBa_K284002 
                 SEQ ID NO: 454 JEN1 Promoter from 
                 . . . gagtaaccaaaaccaaaacagatttcaacc 
               
               
                   
                 
                   Kluyveromyces lactis 
                 
                   
               
               
                   
               
               
                 BBa_K106699 
                 SEQ ID NO: 455 Gal1 Promoter 
                 . . . aaagtaagaatttttgaaaattcaatataa 
               
               
                   
               
               
                 BBa_K165041 
                 SEQ ID NO: 456 Zif268-HIV binding 
                 . . . atacggtcaacgaactataattaactaaac 
               
               
                   
                 sites + TEF constitutive yeast promoter 
                   
               
               
                   
               
               
                 BBa_K165034 
                 SEQ ID NO: 457 Zif268-HIV bs + LexA 
                 . . . cacaaatacacacactaaattaataactag 
               
               
                   
                 bs + mCYC promoter 
                   
               
               
                   
               
               
                 BBa_K165031 
                 SEQ ID NO: 458 mCYC promoter plus 
                 . . . cacaaatacacacactaaattaataactag 
               
               
                   
                 LexA binding sites 
                   
               
               
                   
               
               
                 BBa_K165030 
                 SEQ ID NO: 459 mCYC promoter plus 
                 . . . cacaaatacacacactaaattaataactag 
               
               
                   
                 Zif268-HIV binding sites 
                   
               
               
                   
               
               
                 BBa_K165001 
                 SEQ ID NO: 460 pGAL1 + w/XhoI sites 
                 . . . atactttaacgtcaaggagaaaaaactata 
               
               
                   
               
               
                 BBa_K110016 
                 SEQ ID NO: 461 A-Cell Promoter STE2 
                 . . . accgttaagaaccatatccaagaatcaaaa 
               
               
                   
                 (backwards) 
                   
               
               
                   
               
               
                 BBa_K110015 
                 SEQ ID NO: 462 A-Cell Promoter MFA1 
                 . . . cttcatatataaaccgccagaaatgaatta 
               
               
                   
                 (RtL) 
                   
               
               
                   
               
               
                 BBa_K110014 
                 SEQ ID NO: 463 A-Cell Promoter MFA2 
                 . . . atcttcatacaacaataactaccaacctta 
               
               
                   
                 (backwards) 
                   
               
               
                   
               
               
                 BBa_K110006 
                 SEQ ID NO: 464 Alpha-Cell Promoter 
                 . . . tttcatacacaatataaacgattaaaagaa 
               
               
                   
                 MF(ALPHA)1 
                   
               
               
                   
               
               
                 BBa_K110005 
                 SEQ ID NO: 465 Alpha-Cell Promoter 
                 . . . aaattccagtaaattcacatattggagaaa 
               
               
                   
                 MF(ALPHA)2 
                   
               
               
                   
               
               
                 BBa_K110004 
                 SEQ ID NO: 466 Alpha-Cell Promoter 
                 . . . gggagccagaacgcttctggtggtgtaaat 
               
               
                   
                 Ste3 
                   
               
               
                   
               
               
                 BBa_J24813 
                 SEQ ID NO: 467 URA3 Promoter from  S.   
                 . . . gcacagacttagattggtatatatacgcat 
               
               
                   
                 
                   cerevisiae 
                 
                   
               
               
                   
               
               
                 BBa_K284003 
                 SEQ ID NO: 468 Partial DLD Promoter 
                 . . . aagtgcaagaaagaccagaaacgcaactca 
               
               
                   
                 from  Kluyveromyces lactis   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Examples of Eukaryotic Positive (Activatible) Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I10498 
                 SEQ ID NO: 469 Oct-4 promoter 
                 ...taaaaaaaaaaaaaaaaaaaaaaaaaaaaa 
               
               
                   
               
               
                 BBa_J05215 
                 SEQ ID NO: 470 Regulator for R1- 
                 ... 
               
               
                   
                 CREBH 
                 ggggcgagggccccgcctccggaggcgggg 
               
               
                   
               
               
                 BBa_J05216 
                 SEQ ID NO: 471 Regulator for R3- 
                 ... 
               
               
                   
                 ATF6 
                 gaggggacggctccggccccggggccggag 
               
               
                   
               
               
                 BBa_J05217 
                 SEQ ID NO: 472 Regulator for R2- 
                 ... 
               
               
                   
                 YAP7 
                 ggggcgagggctccggccccggggccggag 
               
               
                   
               
               
                 BBa_J05218 
                 SEQ ID NO: 473 Regulator for R4- 
                 ... 
               
               
                   
                 cMaf 
                 gaggggacggccccgcctccggaggcgggg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Examples of Negatively regulated (repressible)  E. coli  σ70 promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I1051 
                 SEQ ID NO: 474 Lux cassette right promoter 
                 ...tgttatagtcgaatacctctggcggtgata 
               
               
                   
               
               
                 BBa_I12001 
                 SEQ ID NO: 475 Promoter (PRM+) 
                 ... 
               
               
                   
                   
                 gatttaacgtatcagcacaaaaaagaaacc 
               
               
                   
               
               
                 BBa_I12006 
                 SEQ ID NO: 476 Modified lamdba Prm promoter 
                 ...attacaaactttcttgtatagatttaacgt 
               
               
                   
                 (repressed by 434 cI) 
               
               
                   
               
               
                 BBa_I12036 
                 SEQ ID NO: 477 Modified lamdba Prm promoter 
                 ...tttcttgtatagatttacaatgtatcttgt 
               
               
                   
                 (cooperative repression by 434 cI) 
               
               
                   
               
               
                 BBa_I12040 
                 SEQ ID NO: 478 Modified lambda P(RM) 
                 ...tttcttgtagatacttacaatgtatcttgt 
               
               
                   
                 promoter: −10 region from P(L) and 
               
               
                   
                 cooperatively repressed by 434 cI 
               
               
                   
               
               
                 BBa_I12212 
                 SEQ ID NO: 479 TetR - TetR-4C heterodimer 
                 ... 
               
               
                   
                 promoter (negative) 
                 actctgtcaatgatagagtggattcaaaaa 
               
               
                   
               
               
                 BBa_I14015 
                 SEQ ID NO: 480 P(Las) TetO 
                 ...ttttggtacactccctatcagtgatagaga 
               
               
                   
               
               
                 BBa_I14016 
                 SEQ ID NO: 481 P(Las) CIO 
                 ...ctttttggtacactacctctggcggtgata 
               
               
                   
               
               
                 BBa_I14032 
                 SEQ ID NO: 482 promoter P(Lac) IQ 
                 ... 
               
               
                   
                   
                 aaacctttcgcggtatggcatgatagcgcc 
               
               
                   
               
               
                 BBa_I714889 
                 SEQ ID NO: 483 OR21 of PR and PRM 
                 ...tattttacctctggcggtgataatggttgc 
               
               
                   
               
               
                 BBa_I714924 
                 SEQ ID NO: 484 RecA_DlexO_DLacO1 
                 ... 
               
               
                   
                   
                 actctcggcatggacgagctgtacaagtaa 
               
               
                   
               
               
                 BBa_I715003 
                 SEQ ID NO: 485 hybrid pLac with UV5 mutation 
                 ... 
               
               
                   
                   
                 ttgtgagcggataacaatatgttgagcaca 
               
               
                   
               
               
                 BBa_I718018 
                 SEQ ID NO: 486 dapAp promoter 
                 ... 
               
               
                   
                   
                 cattgagacacttgtttgcacagaggatgg 
               
               
                   
               
               
                 BBa_I731004 
                 SEQ ID NO: 487 FecA promoter 
                 ...ttctcgttcgactcatagctgaacacaaca 
               
               
                   
               
               
                 BBa_I732200 
                 SEQ ID NO: 488 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O1wt1) 
                 gaattgtgagcggataacaattggatccgg 
               
               
                   
               
               
                 BBa_I732201 
                 SEQ ID NO: 489 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O11) 
                 ggaattgtgagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732202 
                 SEQ ID NO: 490 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O22) 
                 ggaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732203 
                 SEQ ID NO: 491 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O33) 
                 ggaattgtaaacgtttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732204 
                 SEQ ID NO: 492 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O44) 
                 ggaattgtgaacgttcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732205 
                 SEQ ID NO: 493 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O55) 
                 ggaattttgagcgctcaaaattggatccgg 
               
               
                   
               
               
                 BBa_I732206 
                 SEQ ID NO: 494 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O66) 
                 ggaattatgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732207 
                 SEQ ID NO: 495 NOT Gate Promoter Family 
                 ... 
               
               
                   
                 Member (D001O77) 
                 gggacgactgtatacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732270 
                 SEQ ID NO: 496 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O12) 
                 ggaattgtgagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732271 
                 SEQ ID NO: 497 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O16) 
                 ggaattgtgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732272 
                 SEQ ID NO: 498 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O17) 
                 ggaattgtgagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732273 
                 SEQ ID NO: 499 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O21) 
                 ggaattgtaagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732274 
                 SEQ ID NO: 500 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O24) 
                 ggaattgtaagcgttcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732275 
                 SEQ ID NO: 501 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O26) 
                 ggaattgtaagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732276 
                 SEQ ID NO: 502 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O27) 
                 ggaattgtaagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732277 
                 SEQ ID NO: 503 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O46) 
                 ggaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732278 
                 SEQ ID NO: 504 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O47) 
                 ggaattgtgaactacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732279 
                 SEQ ID NO: 505 Promoter Family Member with 
                 ... 
               
               
                   
                 Hybrid Operator (D001O61) 
                 ggaattatgagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I732301 
                 SEQ ID NO: 506 NAND Candidate 
                 ... 
               
               
                   
                 (U073O26D001O16) 
                 ggaattgtgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732302 
                 SEQ ID NO: 507 NAND Candidate 
                 ... 
               
               
                   
                 (U073O27D001O17) 
                 ggaattgtgagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732303 
                 SEQ ID NO: 508 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D001O46) 
                 ggaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732304 
                 SEQ ID NO: 509 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D001O47) 
                 ggaattgtgaactacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732305 
                 SEQ ID NO: 510 NAND Candidate 
                 ...taaattgtgaacgctcataattggatccgg 
               
               
                   
                 (U073O22D059O46) 
               
               
                   
               
               
                 BBa_I732306 
                 SEQ ID NO: 511 NAND Candidate 
                 ... 
               
               
                   
                 (U073O11D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732351 
                 SEQ ID NO: 512 NOR Candidate 
                 ... 
               
               
                   
                 (U037O11D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732352 
                 SEQ ID NO: 513 NOR Candidate 
                 ... 
               
               
                   
                 (U035O44D001O22) 
                 ggaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732400 
                 SEQ ID NO: 514 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732401 
                 SEQ ID NO: 515 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732402 
                 SEQ ID NO: 516 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732403 
                 SEQ ID NO: 517 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732404 
                 SEQ ID NO: 518 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732405 
                 SEQ ID NO: 519 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732406 
                 SEQ ID NO: 520 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732407 
                 SEQ ID NO: 521 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732408 
                 SEQ ID NO: 522 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U097NUL + D014O22) 
               
               
                   
               
               
                 BBa_I732409 
                 SEQ ID NO: 523 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732410 
                 SEQ ID NO: 524 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U097NUL + D038O22) 
               
               
                   
               
               
                 BBa_I732411 
                 SEQ ID NO: 525 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732412 
                 SEQ ID NO: 526 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732413 
                 SEQ ID NO: 527 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732414 
                 SEQ ID NO: 528 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U097O11 + D014O22) 
               
               
                   
               
               
                 BBa_I732415 
                 SEQ ID NO: 529 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732416 
                 SEQ ID NO: 530 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U097O11 + D038O22) 
               
               
                   
               
               
                 BBa_I732417 
                 SEQ ID NO: 531 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732418 
                 SEQ ID NO: 532 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732419 
                 SEQ ID NO: 533 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732420 
                 SEQ ID NO: 534 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U085O11 + D014O22) 
               
               
                   
               
               
                 BBa_I732421 
                 SEQ ID NO: 535 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732422 
                 SEQ ID NO: 536 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U085O11 + D038O22) 
               
               
                   
               
               
                 BBa_I732423 
                 SEQ ID NO: 537 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732424 
                 SEQ ID NO: 538 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732425 
                 SEQ ID NO: 539 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732426 
                 SEQ ID NO: 540 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U073O11 + D014O22) 
               
               
                   
               
               
                 BBa_I732427 
                 SEQ ID NO: 541 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732428 
                 SEQ ID NO: 542 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U073O11 + D038O22) 
               
               
                   
               
               
                 BBa_I732429 
                 SEQ ID NO: 543 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732430 
                 SEQ ID NO: 544 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732431 
                 SEQ ID NO: 545 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732432 
                 SEQ ID NO: 546 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U061O11 + D014O22) 
               
               
                   
               
               
                 BBa_I732433 
                 SEQ ID NO: 547 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732434 
                 SEQ ID NO: 548 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U061O11 + D038O22) 
               
               
                   
               
               
                 BBa_I732435 
                 SEQ ID NO: 549 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732436 
                 SEQ ID NO: 550 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732437 
                 SEQ ID NO: 551 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732438 
                 SEQ ID NO: 552 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U049O11 + D014O22) 
               
               
                   
               
               
                 BBa_I732439 
                 SEQ ID NO: 553 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732440 
                 SEQ ID NO: 554 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U049O11 + D038O22) 
               
               
                   
               
               
                 BBa_I732441 
                 SEQ ID NO: 555 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732442 
                 SEQ ID NO: 556 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732443 
                 SEQ ID NO: 557 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732444 
                 SEQ ID NO: 558 Promoter Family Member 
                 ...taaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U037O11 + D014O22) 
               
               
                   
               
               
                 BBa_I732445 
                 SEQ ID NO: 559 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732446 
                 SEQ ID NO: 560 Promoter Family Member 
                 ...tcaattgtaagcgcttacaattggatccgg 
               
               
                   
                 (U037O11 + D038O22) 
               
               
                   
               
               
                 BBa_I732447 
                 SEQ ID NO: 561 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732448 
                 SEQ ID NO: 562 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732450 
                 SEQ ID NO: 563 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O26 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732451 
                 SEQ ID NO: 564 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O27 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732452 
                 SEQ ID NO: 565 
                 ... 
               
               
                   
                 (U073O26 + D062O61) Promoter Family Member 
                 caaattatgagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I739101 
                 SEQ ID NO: 566 Double Promoter (constitutive/ 
                 ...tgatagagattccctatcagtgatagagat 
               
               
                   
                 TetR, negative) 
               
               
                   
               
               
                 BBa_I739102 
                 SEQ ID NO: 567 Double Promoter (cI, negative/ 
                 ...tgatagagattccctatcagtgatagagat 
               
               
                   
                 TetR, negative) 
               
               
                   
               
               
                 BBa_I739103 
                 SEQ ID NO: 568 Double Promoter (lacI, negative/ 
                 ...gttctttaattatttaagtgttctttaatt 
               
               
                   
                 P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739104 
                 SEQ ID NO: 569 Double Promoter (LuxR/HSL, 
                 ...gttctttaattatttaagtgttctttaatt 
               
               
                   
                 positive/P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739105 
                 SEQ ID NO: 570 Double Promoter (LuxR/HSL, 
                 ... 
               
               
                   
                 positive/cI, negative) 
                 cgtgcgtgttgataacaccgtgcgtgttga 
               
               
                   
               
               
                 BBa_I739106 
                 SEQ ID NO: 571 Double Promoter (TetR, negative/ 
                 ...gtgttctttaatatttaagtgttctttaat 
               
               
                   
                 P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739107 
                 SEQ ID NO: 572 Double Promoter (cI, negative/ 
                 ... 
               
               
                   
                 LacI, negative) 
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_I746665 
                 SEQ ID NO: 573 Pspac-hy promoter 
                 ...tgtgtgtaattgtgagcggataacaattaa 
               
               
                   
               
               
                 BBa_I751500 
                 SEQ ID NO: 574 pcI (for positive control of pcI- 
                 ...ttttacctctggcggtgataatggttgcag 
               
               
                   
                 lux hybrid promoter) 
               
               
                   
               
               
                 BBa_I751501 
                 SEQ ID NO: 575 plux-cI hybrid promoter 
                 ...gtgttgatgcttttatcaccgccagtggta 
               
               
                   
               
               
                 BBa_I751502 
                 SEQ ID NO: 576 plux-lac hybrid promoter 
                 ... 
               
               
                   
                   
                 agtgtgtggaattgtgagcggataacaatt 
               
               
                   
               
               
                 BBa_I756014 
                 SEQ ID NO: 577 LexAoperator- 
                 ... 
               
               
                   
                 MajorLatePromoter 
                 agggggtgggggcgcgttggcgcgccacac 
               
               
                   
               
               
                 BBa_I761011 
                 SEQ ID NO: 578 CinR, CinL and glucose 
                 ...acatcttaaaagttttagtatcatattcgt 
               
               
                   
                 controlled promoter 
               
               
                   
               
               
                 BBa_J05209 
                 SEQ ID NO: 579 Modified Pr Promoter 
                 ...tattttacctctggcggtgataatggttgc 
               
               
                   
               
               
                 BBa_J05210 
                 SEQ ID NO: 580 Modified Prm + Promoter 
                 ...atttataaatagtggtgatagatttaacgt 
               
               
                   
               
               
                 BBa_J07019 
                 SEQ ID NO: 581 FecA Promoter (with Fur box) 
                 ...acccttctcgttcgactcatagctgaacac 
               
               
                   
               
               
                 BBa_J15301 
                 SEQ ID NO: 582 Pars promoter from  Escherichia   
                 ... 
               
               
                   
                   coli  chromosomal ars operon. 
                 tgacttatccgcttcgaagagagacactac 
               
               
                   
               
               
                 BBa_J22052 
                 SEQ ID NO: 583 Pcya 
                 ...aggtgttaaattgatcacgttttagaccat 
               
               
                   
               
               
                 BBa_J22106 
                 SEQ ID NO: 584 rec A (SOS) Promoter 
                 ...caatttggtaaaggctccatcatgtaataa 
               
               
                   
               
               
                 BBa_J22126 
                 SEQ ID NO: 585 Rec A (SOS) promoter 
                 ... 
               
               
                   
                   
                 gagaaacaatttggtaaaggctccatcatg 
               
               
                   
               
               
                 BBa_J31013 
                 SEQ ID NO: 586 pLac Backwards [cf. 
                 ... 
               
               
                   
                 BBa_R0010] 
                 aacgcgcggggagaggcggtttgcgtattg 
               
               
                   
               
               
                 BBa_J34800 
                 SEQ ID NO: 587 Promoter tetracycline inducible 
                 ... 
               
               
                   
                   
                 cagtgatagagatactgagcacatcagcac 
               
               
                   
               
               
                 BBa_J34806 
                 SEQ ID NO: 588 promoter lac induced 
                 ...ttatgcttccggctcgtataatgtttcaaa 
               
               
                   
               
               
                 BBa_J34809 
                 SEQ ID NO: 589 promoter lac induced 
                 ... 
               
               
                   
                   
                 ggctcgtatgttgtgtcgaccgagctgcgc 
               
               
                   
               
               
                 BBa_J54016 
                 SEQ ID NO: 590 promoter_lacq 
                 ... 
               
               
                   
                   
                 aaacctttcgcggtatggcatgatagcgcc 
               
               
                   
               
               
                 BBa_J54120 
                 SEQ ID NO: 591 EmrR_regulated promoter 
                 ...atttgtcactgtcgttactatatcggctgc 
               
               
                   
               
               
                 BBa_J54130 
                 SEQ ID NO: 592 BetI_regulated promoter 
                 ...gtccaatcaataaccgctttaatagataaa 
               
               
                   
               
               
                 BBa_J56012 
                 SEQ ID NO: 593 Invertible sequence of dna 
                 ...actttattatcaataagttaaatcggtacc 
               
               
                   
                 includes Ptrc promoter 
               
               
                   
               
               
                 BBa_J64065 
                 SEQ ID NO: 594 cI repressed promoter 
                 ...gtgttgactattttacctctggcggtgata 
               
               
                   
               
               
                 BBa_J64067 
                 SEQ ID NO: 595 LuxR + 3OC6HSL independent 
                 ...gtgttgactattttacctctggcggtgata 
               
               
                   
                 R0065 
               
               
                   
               
               
                 BBa_J64068 
                 SEQ ID NO: 596 increased strength R0051 
                 ...atacctctggcggtgatatataatggttgc 
               
               
                   
               
               
                 BBa_J64069 
                 SEQ ID NO: 597 R0065 with lux box deleted 
                 ...gtgttgactattttacctctggcggtgata 
               
               
                   
               
               
                 BBa_J64712 
                 SEQ ID NO: 598 LasR/LasI Inducible &amp; 
                 ... 
               
               
                   
                 RHLR/RHLI repressible Promoter 
                 gaaatctggcagtttttggtacacgaaagc 
               
               
                   
               
               
                 BBa_J64800 
                 SEQ ID NO: 599 RHLR/RHLI Inducible &amp; 
                 ... 
               
               
                   
                 LasR/LasI repressible Promoter 
                 tgccagttctggcaggtctaaaaagtgttc 
               
               
                   
               
               
                 BBa_J64981 
                 SEQ ID NO: 600 OmpR-P strong binding, 
                 ...agcgctcacaatttaatacgactcactata 
               
               
                   
                 regulatory region for Team Challenge03-2007 
               
               
                   
               
               
                 BBa_J64987 
                 SEQ ID NO: 601 LacI Consensus Binding Site in 
                 ...taataattgtgagcgctcacaattttgaca 
               
               
                   
                 sigma 70 binding region 
               
               
                   
               
               
                 BBa_J72005 
                 SEQ ID NO: 602 {Piet} promoter in BBb 
                 ... 
               
               
                   
                   
                 atccctatcagtgatagagatactgagcac 
               
               
                   
               
               
                 BBa_K086017 
                 SEQ ID NO: 603 unmodified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter 
                 ttgtgagcggataacaagatactgagcaca 
               
               
                   
               
               
                 BBa_K091100 
                 SEQ ID NO: 604 pLac_lux hybrid promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_K091101 
                 SEQ ID NO: 605 pTet_Lac hybrid promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_K091104 
                 SEQ ID NO: 606 pLac/Mnt Hybrid Promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_K091105 
                 SEQ ID NO: 607 pTet/Mnt Hybrid Promoter 
                 ... 
               
               
                   
                   
                 agaactgtaatccctatcagtgatagagat 
               
               
                   
               
               
                 BBa_K091106 
                 SEQ ID NO: 608 LsrA/cI hybrid promoter 
                 ...tgttgatttatctaacaccgtgcgtgttga 
               
               
                   
               
               
                 BBa_K091107 
                 SEQ ID NO: 609 pLux/cI Hybrid Promoter 
                 ... 
               
               
                   
                   
                 acaccgtgcgtgttgatatagtcgaataaa 
               
               
                   
               
               
                 BBa_K091110 
                 SEQ ID NO: 610 LacI Promoter 
                 ... 
               
               
                   
                   
                 cctttcgcggtatggcatgatagcgcccgg 
               
               
                   
               
               
                 BBa_K091111 
                 SEQ ID NO: 611 LacIQ promoter 
                 ... 
               
               
                   
                   
                 cctttcgcggtatggcatgatagcgcccgg 
               
               
                   
               
               
                 BBa_K091112 
                 SEQ ID NO: 612 pLacIQ1 promoter 
                 ... 
               
               
                   
                   
                 cctttcgcggtatggcatgatagcgcccgg 
               
               
                   
               
               
                 BBa_K091143 
                 SEQ ID NO: 613 pLas/cI Hybrid Promoter 
                 ...ggttatttttggtacctctggcggtgataa 
               
               
                   
               
               
                 BBa_K091146 
                 SEQ ID NO: 614 pLas/Lux Hybrid Promoter 
                 ...tgtaggatcgtacaggtataaattcttcag 
               
               
                   
               
               
                 BBa_K091157 
                 SEQ ID NO: 615 pLux/Las Hybrid Promoter 
                 ...ctatctcatttgctagtatagtcgaataaa 
               
               
                   
               
               
                 BBa_K093000 
                 SEQ ID NO: 616 pRecA with LexA binding site 
                 ...gtatatatatacagtataattgcttcaaca 
               
               
                   
               
               
                 BBa_K093008 
                 SEQ ID NO: 617 reverse BBa_R0011 
                 ...cacaatgtcaattgttatccgctcacaatt 
               
               
                   
               
               
                 BBa_K094120 
                 SEQ ID NO: 618 pLacI/ara-1 
                 ... 
               
               
                   
                   
                 aattgtgagcggataacaatttcacacaga 
               
               
                   
               
               
                 BBa_K094140 
                 SEQ ID NO: 619 pLacIq 
                 ... 
               
               
                   
                   
                 ccggaagagagtcaattcagggtggtgaat 
               
               
                   
               
               
                 BBa_K101000 
                 SEQ ID NO: 620 Dual-Repressed Promoter for 
                 ... 
               
               
                   
                 p22 mnt and TetR 
                 acggtgacctagatctccgatactgagcac 
               
               
                   
               
               
                 BBa_K101001 
                 SEQ ID NO: 621 Dual-Repressed Promoter for 
                 ... 
               
               
                   
                 LacI and LambdacI 
                 tggaattgtgagcggataaaatttcacaca 
               
               
                   
               
               
                 BBa_K101002 
                 SEQ ID NO: 622 Dual-Repressed Promoter for 
                 ...tagtagataatttaagtgttctttaatttc 
               
               
                   
                 p22 cII and TetR 
               
               
                   
               
               
                 BBa_K101017 
                 SEQ ID NO: 623 MioC Promoter (DNAa- 
                 ... 
               
               
                   
                 Repressed Promoter) 
                 ccaacgcgttcacagcgtacaattactagt 
               
               
                   
               
               
                 BBa_K109200 
                 SEQ ID NO: 624 AraC and TetR promoter 
                 ... 
               
               
                   
                 (hybrid) 
                 aacaaaaaaacggatcctctagttgcggcc 
               
               
                   
               
               
                 BBa_K112118 
                 SEQ ID NO: 625 rrnB P1 promoter 
                 ... 
               
               
                   
                   
                 ataaatgcttgactctgtagcgggaaggcg 
               
               
                   
               
               
                 BBa_K112318 
                 SEQ ID NO: 626 {&lt;bolA promoter&gt;} in BBb 
                 ... 
               
               
                   
                 format 
                 atttcatgatgatacgtgagcggatagaag 
               
               
                   
               
               
                 BBa_K112401 
                 SEQ ID NO: 627 Promoter for recA gene - SOS 
                 ... 
               
               
                   
                 and Ultrasound Sensitive 
                 caaacagaaagcgttggcggcagcactggg 
               
               
                   
               
               
                 BBa_K112402 
                 SEQ ID NO: 628 promoter for FabA gene - 
                 ... 
               
               
                   
                 Membrane Damage and Ultrasound Sensitive 
                 gtcaaaatgaccgaaacgggtggtaacttc 
               
               
                   
               
               
                 BBa_K112405 
                 SEQ ID NO: 629 Promoter for CadA and CadB 
                 ...agtaatcttatcgccagtttggtctggtca 
               
               
                   
                 genes 
               
               
                   
               
               
                 BBa_K112406 
                 SEQ ID NO: 630 cadC promoter 
                 ...agtaatcttatcgccagtttggtctggtca 
               
               
                   
               
               
                 BBa_K112701 
                 SEQ ID NO: 631 hns promoter 
                 ...aattctgaacaacatccgtactcttcgtgc 
               
               
                   
               
               
                 BBa_K112708 
                 SEQ ID NO: 632 PfhuA 
                 ...tttacgttatcattcactttacatcagagt 
               
               
                   
               
               
                 BBa_K113009 
                 SEQ ID NO: 633 pBad/araC 
                 ...gtttctccatacccgtttttttgggctagc 
               
               
                   
               
               
                 BBa_K116001 
                 SEQ ID NO: 634 nhaA promoter that can be 
                 ... 
               
               
                   
                 regulated by pH and nhaR protein. 
                 cgatctattcacctgaaagagaaataaaaa 
               
               
                   
               
               
                 BBa_K116500 
                 SEQ ID NO: 635 OmpF promoter that is activated 
                 ... 
               
               
                   
                 or repressed by OmpR according to osmolarity. 
                 aaacgttagtttgaatggaaagatgcctgc 
               
               
                   
               
               
                 BBa_K119002 
                 SEQ ID NO: 636 RcnR operator (represses RcnA) 
                 ...attgccgaattaatactaagaattattatc 
               
               
                   
               
               
                 BBa_K121011 
                 SEQ ID NO: 637 promoter (lacI regulated) 
                 ... 
               
               
                   
                   
                 acaggaaacagctatgaccatgattacgcc 
               
               
                   
               
               
                 BBa_K121014 
                 SEQ ID NO: 638 promoter (lambda cI regulated) 
                 ... 
               
               
                   
                   
                 actggcggttataatgagcacatcagcagg 
               
               
                   
               
               
                 BBa_K137046 
                 SEQ ID NO: 639 150 bp inverted tetR promoter 
                 ... 
               
               
                   
                   
                 caccgacaaacaacagataaaacgaaaggc 
               
               
                   
               
               
                 BBa_K137047 
                 SEQ ID NO: 640 250 bp inverted tetR promoter 
                 ...agtgttattaagctactaaagcgtagtttt 
               
               
                   
               
               
                 BBa_K137048 
                 SEQ ID NO: 641 350 bp inverted tetR promoter 
                 ... 
               
               
                   
                   
                 gaataagaaggctggctctgcaccttggtg 
               
               
                   
               
               
                 BBa_K137049 
                 SEQ ID NO: 642 450 bp inverted tetR promoter 
                 ...ttagcgacttgatgctcttgatcttccaat 
               
               
                   
               
               
                 BBa_K137050 
                 SEQ ID NO: 643 650 bp inverted tetR promoter 
                 ...acatctaaaacttttagcgttattacgtaa 
               
               
                   
               
               
                 BBa_K137051 
                 SEQ ID NO: 644 850 bp inverted tetR promoter 
                 ... 
               
               
                   
                   
                 ttccgacctcattaagcagctctaatgcgc 
               
               
                   
               
               
                 BBa_K137124 
                 SEQ ID NO: 645 LacI-repressed promoter A81 
                 ...caatttttaaacctgtaggatcgtacaggt 
               
               
                   
               
               
                 BBa_K137125 
                 SEQ ID NO: 646 LacI-repressed promoter B4 
                 ...caatttttaaaattaaaggcgttacccaac 
               
               
                   
               
               
                 BBa_K145150 
                 SEQ ID NO: 647 Hybrid promoter: HSL-LuxR 
                 ...tagtttataatttaagtgttctttaatttc 
               
               
                   
                 activated, P22 C2 repressed 
               
               
                   
               
               
                 BBa_K145152 
                 SEQ ID NO: 648 Hybrid promoter: P22 c2, LacI 
                 ... 
               
               
                   
                 NOR gate 
                 gaaaatgtgagcgagtaacaacctcacaca 
               
               
                   
               
               
                 BBa_K256028 
                 SEQ ID NO: 649 placI: CHE 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K259005 
                 SEQ ID NO: 650 AraC Rheostat Promoter 
                 ...ttttatcgcaactctctactgtttctccat 
               
               
                   
               
               
                 BBa_K259007 
                 SEQ ID NO: 651 AraC Promoter fused with RBS 
                 ... 
               
               
                   
                   
                 gtttctccattactagagaaagaggggaca 
               
               
                   
               
               
                 BBa_K266001 
                 SEQ ID NO: 652 Inverter TetR -&gt; LuxR 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K266003 
                 SEQ ID NO: 653 POPS -&gt; Lac Inverter -&gt; LasR 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K266004 
                 SEQ ID NO: 654 Const Lac Inverter -&gt; LasR 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K266005 
                 SEQ ID NO: 655 PAI + LasR -&gt; LasI &amp; AI + LuxR--| 
                 ...aataactctgatagtgctagtgtagatctc 
               
               
                   
                 LasI 
               
               
                   
               
               
                 BBa_K266006 
                 SEQ ID NO: 656 PAI + LasR -&gt; LasI + GFP &amp; 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 AI + LuxR--|LasI + GFP 
               
               
                   
               
               
                 BBa_K266007 
                 SEQ ID NO: 657 Complex QS -&gt; LuxI &amp; LasI 
                 ...caccttcgggtgggcctttctgcgtttata 
               
               
                   
                 circuit 
               
               
                   
               
               
                 BBa_K266008 
                 SEQ ID NO: 658 J23100 + Lac inverter 
                 ... 
               
               
                   
                   
                 ttgtgagcggataacaagatactgagcaca 
               
               
                   
               
               
                 BBa_K266009 
                 SEQ ID NO: 659 J23100 + Lac inverter + RBS 
                 ... 
               
               
                   
                   
                 actgagcacatactagagaaagaggagaaa 
               
               
                   
               
               
                 BBa_K266011 
                 SEQ ID NO: 660 Lac Inverter and strong RBS 
                 ... 
               
               
                   
                   
                 actgagcacatactagagaaagaggagaaa 
               
               
                   
               
               
                 BBa_K292002 
                 SEQ ID NO: 661 pLac (LacI regulated) + Strong 
                 ... 
               
               
                   
                 RBS 
                 tcacacatactagagattaaagaggagaaa 
               
               
                   
               
               
                 BBa_M31370 
                 SEQ ID NO: 662 tacI Promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_R0010 
                 SEQ ID NO: 663 promoter (lacI regulated) 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_R0011 
                 SEQ ID NO: 664 Promoter (lacI regulated, lambda 
                 ... 
               
               
                   
                 pL hybrid) 
                 ttgtgagcggataacaagatactgagcaca 
               
               
                   
               
               
                 BBa_R0040 
                 SEQ ID NO: 665 TetR repressible promoter 
                 ... 
               
               
                   
                   
                 atccctatcagtgatagagatactgagcac 
               
               
                   
               
               
                 BBa_R0050 
                 SEQ ID NO: 666 Promoter (HK022 cI regulated) 
                 ... 
               
               
                   
                   
                 ccgtcataatatgaaccataagttcaccac 
               
               
                   
               
               
                 BBa_R0051 
                 SEQ ID NO: 667 promoter (lambda cI regulated) 
                 ...tattttacctctggcggtgataatggttgc 
               
               
                   
               
               
                 BBa_R0052 
                 SEQ ID NO: 668 Promoter (434 cI regulated) 
                 ...attgtatgaaaatacaagaaagtttgttga 
               
               
                   
               
               
                 BBa_R0053 
                 SEQ ID NO: 669 Promoter (p22 cII regulated) 
                 ...tagtagataatttaagtgttattaatttc 
               
               
                   
               
               
                 BBa_R0061 
                 SEQ ID NO: 670 Promoter (HSL-mediated luxR 
                 ttgacacctgtaggatcgtacaggtataat 
               
               
                   
                 repressor) 
               
               
                   
               
               
                 BBa_R0063 
                 SEQ ID NO: 671 Promoter (luxR &amp; HSL regulated -- 
                 ... 
               
               
                   
                 lux pL) 
                 cacgcaaaacttgcgacaaacaataggtaa 
               
               
                   
               
               
                 BBa_R0065 
                 SEQ ID NO: 672 Promoter (lambda cI and luxR 
                 ...gtgttgactattttacctctggcggtgata 
               
               
                   
                 regulated -- hybrid) 
               
               
                   
               
               
                 BBa_R0073 
                 SEQ ID NO: 673 Promoter (Mnt regulated) 
                 ...tagatctcctatagtgagtcgtattaattt 
               
               
                   
               
               
                 BBa_R0074 
                 SEQ ID NO: 674 Promoter (PenI regulated) 
                 ...tactttcaaagactacatttgtaagatttg 
               
               
                   
               
               
                 BBa_R0075 
                 SEQ ID NO: 675 Promoter (TP901 cI regulated) 
                 ... 
               
               
                   
                   
                 cataaagttcatgaaacgtgaactgaaatt 
               
               
                   
               
               
                 BBa_R1050 
                 SEQ ID NO: 676 Promoter, Standard (HK022 cI 
                 ... 
               
               
                   
                 regulated) 
                 ccgtgatactatgaaccataagttcaccac 
               
               
                   
               
               
                 BBa_R1051 
                 SEQ ID NO: 677 Promoter, Standard (lambda cI 
                 ...aattttacctctggcggtgatactggttgc 
               
               
                   
                 regulated) 
               
               
                   
               
               
                 BBa_R1052 
                 SEQ ID NO: 678 Promoter, Standard (434 cI 
                 ...attgtatgatactacaagaaagtttgttga 
               
               
                   
                 regulated) 
               
               
                   
               
               
                 BBa_R1053 
                 SEQ ID NO: 679 Promoter, Standard (p22 cII 
                 ...tagtagatactttaagtgttctttaatttc 
               
               
                   
                 regulated) 
               
               
                   
               
               
                 BBa_R2000 
                 SEQ ID NO: 680 Promoter, Zif23 regulated, test: 
                 ... 
               
               
                   
                 between 
                 tggtcccacgcgcgtgggatactacgtcag 
               
               
                   
               
               
                 BBa_R2001 
                 SEQ ID NO: 681 Promoter, Zif23 regulated, test: 
                 ... 
               
               
                   
                 after 
                 attacggtgagatactcccacgcgcgtggg 
               
               
                   
               
               
                 BBa_R2002 
                 SEQ ID NO: 682 Promoter, Zif23 regulated, test: 
                 ... 
               
               
                   
                 between and after 
                 acgcgcgtgggatactcccacgcgcgtggg 
               
               
                   
               
               
                 BBa_R2108 
                 SEQ ID NO: 683 Promoter with operator site for 
                 ...gattagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2109 
                 SEQ ID NO: 684 Promoter with operator site for 
                 ...acttagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2110 
                 SEQ ID NO: 685 Promoter with operator site for 
                 ...ggttagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2111 
                 SEQ ID NO: 686 Promoter with operator site for 
                 ...acttagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2112 
                 SEQ ID NO: 687 Promoter with operator site for 
                 ...aattagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2113 
                 SEQ ID NO: 688 Promoter with operator site for 
                 ...acttagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2114 
                 SEQ ID NO: 689 Promoter with operator site for 
                 ...atttagattcataaatttgagagaggagtt 
               
               
                   
                 C2003 
               
               
                   
               
               
                 BBa_R2201 
                 SEQ ID NO: 690 C2006-repressible promoter 
                 ... 
               
               
                   
                   
                 cacgcgcgtgggaatgttataatacgtcag 
               
               
                   
               
               
                 BBa_S04209 
                 SEQ ID NO: 691 R0051:Q04121:B0034:C0079:B0015 
                 ... 
               
               
                   
                   
                 actgagcacatactagagaaagaggagaaa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Examples of Negatively regulated (repressible)  E. coli  σ s  promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K086030 
                 SEQ ID NO: 692 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ38 
                 cagtgagcgagtaacaactacgctgtttta 
               
               
                   
               
               
                 BBa_K086031 
                 SEQ ID NO: 693 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ38 
                 cagtgagcgagtaacaactacgctgtttta 
               
               
                   
               
               
                 BBa_K086032 
                 SEQ ID NO: 694 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ38 
                 atgtgagcggataacactataattaataga 
               
               
                   
               
               
                 BBa_K086033 
                 SEQ ID NO: 695 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ38 
                 atgtgagcggataacactataattaataga 
               
               
                   
               
               
                 BBa_K112318 
                 SEQ ID NO: 696 {&lt;bolA promoter&gt;} in 
                 ... 
               
               
                   
                 BBb format 
                 atttcatgatgatacgtgagcggatagaag 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Examples of Negatively regulated (repressible)  E. coli  σ32 promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K086026 
                 SEQ ID NO: 697 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtggcaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086027 
                 SEQ ID NO: 698 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtgacaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086028 
                 SEQ ID NO: 699 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtaacaccattaagtacgta 
               
               
                   
               
               
                 BBa_K086029 
                 SEQ ID NO: 700 modified Lutz-Bujard LacO 
                 ... 
               
               
                   
                 promoter, with alternative sigma factor σ32 
                 ttgtgagcgagtaacaccattaagtacgta 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Examples of Negatively regulated (repressible) 
               
               
                   E. coli  σ54 promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J64979 
                 SEQ ID NO: 701 glnAp2 
                 ...agttggcacagatttcgctttatctttttt 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Examples of Repressible  B. subtilis  σ A  promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K090501 
                 SEQ ID NO: 702 Gram-Positive IPTG- 
                 ... 
               
               
                   
                 Inducible Promoter 
                 tggaattgtgagcggataacaattaagctt 
               
               
                   
               
               
                 BBa_K143014 
                 SEQ ID NO: 703 Promoter Xyl for  B. subtilis   
                 ... 
               
               
                   
                   
                 agtttgtttaaacaacaaactaataggtga 
               
               
                   
               
               
                 BBa_K143015 
                 SEQ ID NO: 704 Promoter hyper-spank for 
                 ... 
               
               
                   
                 
                   B. subtilis 
                 
                 aatgtgtgtaattgtgagcggataacaatt 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Examples of T7 Repressible Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_R0184 
                 SEQ ID NO: 705 T7 promoter (lacI 
                 .. 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_R0185 
                 SEQ ID NO: 706 T7 promoter (lacI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_R0186 
                 SEQ ID NO: 707 T7 promoter (lacI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
               
                 BBa_R0187 
                 SEQ ID NO: 708 T7 promoter (lacI 
                 ... 
               
               
                   
                 repressible) 
                 ataggggaattgtgagcggataacaattcc 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Examples of Yeast Repressible Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I766558 
                 SEQ ID NO: 709 pFig1 (Inducible) 
                 ... 
               
               
                   
                 Promoter 
                 aaacaaacaaacaaaaaaaaaaaaaaaaaa 
               
               
                   
               
               
                 BBa_I766214 
                 SEQ ID NO: 710 pGal1 
                 ...atactttaacgtcaaggagaaaaaactata 
               
               
                   
               
               
                 BBa_K165000 
                 SEQ ID NO: 711 MET 25 Promoter 
                 ...tagatacaattctattacccccatccatac 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Examples of Eukaryotic Repressible Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I756015 
                 SEQ ID NO: 712 CMV Promoter with lac 
                 ...ttagtgaaccgtcagatcactagtctgcag 
               
               
                   
                 operator sites 
               
               
                   
               
               
                 BBa_I756016 
                 SEQ ID NO: 713 CMV-tet promoter 
                 ...ttagtgaaccgtcagatcactagtctgcag 
               
               
                   
               
               
                 BBa_I756017 
                 SEQ ID NO: 714 U6 promoter with tet 
                 ... 
               
               
                   
                 operators 
                 ggaaaggacgaaacaccgactagtctgcag 
               
               
                   
               
               
                 BBa_I756018 
                 SEQ ID NO: 715 Lambda Operator in SV- 
                 ...attgtttgtgtattttagactagtctgcag 
               
               
                   
                 40 intron 
               
               
                   
               
               
                 BBa_I756019 
                 SEQ ID NO: 716 Lac Operator in SV-40 
                 ...attgtttgtgtattttagactagtctgcag 
               
               
                   
                 intron 
               
               
                   
               
               
                 BBa_I756020 
                 SEQ ID NO: 717 Tet Operator in SV-40 
                 ...attgtttgtgtattttagactagtctgcag 
               
               
                   
                 intron 
               
               
                   
               
               
                 BBa_I756021 
                 SEQ ID NO: 718 CMV promoter with 
                 ...ttagtgaaccgtcagatcactagtctgcag 
               
               
                   
                 Lambda Operator 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Examples of Combination Inducible &amp; Repressible  E. coli  Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I1051 
                 SEQ ID NO: 719 Lux cassette right promoter 
                 ... 
               
               
                   
                   
                 tgttatagtcgaatacctctggcggtgata 
               
               
                   
               
               
                 BBa_I12006 
                 SEQ ID NO: 720 Modified lambda Prm 
                 ...attacaaactttcttgtatagatttaacgt 
               
               
                   
                 promoter (repressed by 434 cI) 
               
               
                   
               
               
                 BBa_I12036 
                 SEQ ID NO: 721 Modified lambda Prm 
                 ...tttcttgtatagatttacaatgtatcttgt 
               
               
                   
                 promoter (cooperative repression by 434 cI) 
               
               
                   
               
               
                 BBa_I12040 
                 SEQ ID NO: 722 modified lambda P(RM) 
                 ...tttcttgtagatacttacaatgtatcttgt 
               
               
                   
                 promoter: −10 region from P(L) and cooperatively 
               
               
                   
                 repressed by 434 cI 
               
               
                   
               
               
                 BBa_I14015 
                 SEQ ID NO: 723 P(Las) TetO 
                 ... 
               
               
                   
                   
                 ttttggtacactccctatcagtgatagaga 
               
               
                   
               
               
                 BBa_I14016 
                 SEQ ID NO: 724 P(Las) CIO 
                 ... 
               
               
                   
                   
                 ctttttggtacactacctctggcggtgata 
               
               
                   
               
               
                 BBa_I714924 
                 SEQ ID NO: 725 RecA DlexO_DLacO1 
                 ... 
               
               
                   
                   
                 actctcggcatggacgagctgtacaagtaa 
               
               
                   
               
               
                 BBa_I731004 
                 SEQ ID NO: 726 FecA promoter 
                 ... 
               
               
                   
                   
                 ttctcgttcgactcatagctgaacacaaca 
               
               
                   
               
               
                 BBa_I732301 
                 SEQ ID NO: 727 NAND Candidate 
                 ... 
               
               
                   
                 (U073O26D001O16) 
                 ggaattgtgagcgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732302 
                 SEQ ID NO: 728 NAND Candidate 
                 ... 
               
               
                   
                 (U073O27D001O17) 
                 ggaattgtgagctacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732303 
                 SEQ ID NO: 729 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D001O46) 
                 ggaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732304 
                 SEQ ID NO: 730 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D001O47) 
                 ggaattgtgaactacagtcgtcggatccgg 
               
               
                   
               
               
                 BBa_I732305 
                 SEQ ID NO: 731 NAND Candidate 
                 ... 
               
               
                   
                 (U073O22D059O46) 
                 taaattgtgaacgctcataattggatccgg 
               
               
                   
               
               
                 BBa_I732306 
                 SEQ ID NO: 732 NAND Candidate 
                 ... 
               
               
                   
                 (U073O11D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732351 
                 SEQ ID NO: 733 NOR Candidate 
                 ... 
               
               
                   
                 (U037O11D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732352 
                 SEQ ID NO: 734 NOR Candidate 
                 ... 
               
               
                   
                 (U035O44D001O22) 
                 ggaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732400 
                 SEQ ID NO: 735 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732401 
                 SEQ ID NO: 736 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732402 
                 SEQ ID NO: 737 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732403 
                 SEQ ID NO: 738 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732404 
                 SEQ ID NO: 739 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732405 
                 SEQ ID NO: 740 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732406 
                 SEQ ID NO: 741 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732407 
                 SEQ ID NO: 742 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732408 
                 SEQ ID NO: 743 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732409 
                 SEQ ID NO: 744 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732410 
                 SEQ ID NO: 745 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732411 
                 SEQ ID NO: 746 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732412 
                 SEQ ID NO: 747 Promoter Family Member 
                 ... 
               
               
                   
                 (U097NUL + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732413 
                 SEQ ID NO: 748 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732414 
                 SEQ ID NO: 749 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732415 
                 SEQ ID NO: 750 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732416 
                 SEQ ID NO: 751 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732417 
                 SEQ ID NO: 752 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732418 
                 SEQ ID NO: 753 Promoter Family Member 
                 ... 
               
               
                   
                 (U097O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732419 
                 SEQ ID NO: 754 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732420 
                 SEQ ID NO: 755 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732421 
                 SEQ ID NO: 756 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732422 
                 SEQ ID NO: 757 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732423 
                 SEQ ID NO: 758 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732424 
                 SEQ ID NO: 759 Promoter Family Member 
                 ... 
               
               
                   
                 (U085O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732425 
                 SEQ ID NO: 760 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732426 
                 SEQ ID NO: 761 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732427 
                 SEQ ID NO: 762 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732428 
                 SEQ ID NO: 763 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732429 
                 SEQ ID NO: 764 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732430 
                 SEQ ID NO: 765 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732431 
                 SEQ ID NO: 766 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732432 
                 SEQ ID NO: 767 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732433 
                 SEQ ID NO: 768 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732434 
                 SEQ ID NO: 769 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732435 
                 SEQ ID NO: 770 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732436 
                 SEQ ID NO: 771 Promoter Family Member 
                 ... 
               
               
                   
                 (U061O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732437 
                 SEQ ID NO: 772 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732438 
                 SEQ ID NO: 773 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11+D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732439 
                 SEQ ID NO: 774 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732440 
                 SEQ ID NO: 775 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732441 
                 SEQ ID NO: 776 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732442 
                 SEQ ID NO: 777 Promoter Family Member 
                 ... 
               
               
                   
                 (U049O11 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732443 
                 SEQ ID NO: 778 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D002O22) 
                 gaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732444 
                 SEQ ID NO: 779 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D014O22) 
                 taaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732445 
                 SEQ ID NO: 780 Promoter Family Member 
                 ... 
               
               
                   
                 (U037011 + D026O22) 
                 gtaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732446 
                 SEQ ID NO: 781 Promoter Family Member 
                 ... 
               
               
                   
                 (U037011 + D038O22) 
                 tcaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732447 
                 SEQ ID NO: 782 Promoter Family Member 
                 ... 
               
               
                   
                 (U037O11 + D050O22) 
                 aaaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732448 
                 SEQ ID NO: 783 Promoter Family Member 
                 ... 
               
               
                   
                 (U037011 + D062O22) 
                 caaattgtaagcgcttacaattggatccgg 
               
               
                   
               
               
                 BBa_I732450 
                 SEQ ID NO: 784 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O26 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732451 
                 SEQ ID NO: 785 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O27 + D062NUL) 
                 gccaaattaaacaggattaacaggatccgg 
               
               
                   
               
               
                 BBa_I732452 
                 SEQ ID NO: 786 Promoter Family Member 
                 ... 
               
               
                   
                 (U073O26 + D062O61) 
                 caaattatgagcgctcacaattggatccgg 
               
               
                   
               
               
                 BBa_I739102 
                 SEQ ID NO: 787 Double Promoter (cI, negative/ 
                 ... 
               
               
                   
                 TetR, negative) 
                 tgatagagattccctatcagtgatagagat 
               
               
                   
               
               
                 BBa_I739103 
                 SEQ ID NO: 788 Double Promoter (lacI, 
                 ...gttctttaattatttaagtgttctttaatt 
               
               
                   
                 negative/P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739104 
                 SEQ ID NO: 789 Double Promoter (LuxR/HSL, 
                 ...gttctttaattatttaagtgttctttaatt 
               
               
                   
                 positive/P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739105 
                 SEQ ID NO: 790 Double Promoter (LuxR/HSL, 
                 ... 
               
               
                   
                 positive/cI, negative) 
                 cgtgcgtgttgataacaccgtgcgtgttga 
               
               
                   
               
               
                 BBa_I739106 
                 SEQ ID NO: 791 Double Promoter (TetR, 
                 ...gtgttctttaatatttaagtgttctttaat 
               
               
                   
                 negative/P22 cII, negative) 
               
               
                   
               
               
                 BBa_I739107 
                 SEQ ID NO: 792 Double Promoter (cI, negative/ 
                 ... 
               
               
                   
                 LacI, negative) 
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_I741018 
                 SEQ ID NO: 793 Right facing promoter (for 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 xylF) controlled by xylR and CRP-cAMP 
               
               
                   
               
               
                 BBa_I741019 
                 SEQ ID NO: 794 Right facing promoter (for 
                 ... 
               
               
                   
                 xylA) controlled by xylR and CRP-cAMP 
                 gcaaaataaaatggaatgatgaaactgggt 
               
               
                   
               
               
                 BBa_I742124 
                 SEQ ID NO: 795 Reverse complement Lac 
                 ... 
               
               
                   
                 promoter 
                 aacgcgcggggagaggcggtttgcgtattg 
               
               
                   
               
               
                 BBa_I751501 
                 SEQ ID NO: 796 plux-cI hybrid promoter 
                 ... 
               
               
                   
                   
                 gtgttgatgcttttatcaccgccagtggta 
               
               
                   
               
               
                 BBa_I751502 
                 SEQ ID NO: 797 plux-lac hybrid promoter 
                 ... 
               
               
                   
                   
                 agtgtgtggaattgtgagcggataacaatt 
               
               
                   
               
               
                 BBa_I761011 
                 SEQ ID NO: 798 CinR, CinL and glucose 
                 ...acatcttaaaagttttagtatcatattcgt 
               
               
                   
                 controlled promoter 
               
               
                   
               
               
                 BBa_I765007 
                 SEQ ID NO: 799 Fe and UV promoters 
                 ... 
               
               
                   
                   
                 ctgaaagcgcataccgctatggagggggtt 
               
               
                   
               
               
                 BBa_J05209 
                 SEQ ID NO: 800 Modified Pr Promoter 
                 ... 
               
               
                   
                   
                 tattttacctctggcggtgataatggttgc 
               
               
                   
               
               
                 BBa_J05210 
                 SEQ ID NO: 801 Modified Prm + Promoter 
                 ...atttataaatagtggtgatagatttaacgt 
               
               
                   
               
               
                 BBa_J58100 
                 SEQ ID NO: 802 AND-type promoter 
                 ...atttataaatagtggtgatagatttaacgt 
               
               
                   
                 synergistically activated by cI and CRP 
               
               
                   
               
               
                 BBa_J64712 
                 SEQ ID NO: 803 LasR/LasI Inducible &amp; 
                 ... 
               
               
                   
                 RHLR/RHLI repressible Promoter 
                 gaaatctggcagtttttggtacacgaaagc 
               
               
                   
               
               
                 BBa_J64800 
                 SEQ ID NO: 804 RHLR/RHLI Inducible &amp; 
                 ... 
               
               
                   
                 LasR/LasI repressible Promoter 
                 tgccagttctggcaggtctaaaaagtgttc 
               
               
                   
               
               
                 BBa_J64804 
                 SEQ ID NO: 805 The promoter region 
                 ... 
               
               
                   
                 (inclusive of regulator binding sites) of the B. subtilis 
                 cacagaacttgcatttatataaagggaaag 
               
               
                   
                 RocDEF operon 
               
               
                   
               
               
                 BBa_J64979 
                 SEQ ID NO: 806 glnAp2 
                 ...agttggcacagatttcgctttatctttttt 
               
               
                   
               
               
                 BBa_J64981 
                 SEQ ID NO: 807 OmpR-P strong binding, 
                 ... 
               
               
                   
                 regulatory region for Team Challenge03-2007 
                 agcgctcacaatttaatacgactcactata 
               
               
                   
               
               
                 BBa_K091100 
                 SEQ ID NO: 808 pLac_lux hybrid promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_K091101 
                 SEQ ID NO: 809 pTet_Lac hybrid promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_K091104 
                 SEQ ID NO: 810 pLac/Mnt Hybrid Promoter 
                 ... 
               
               
                   
                   
                 ggaattgtgagcggataacaatttcacaca 
               
               
                   
               
               
                 BBa_K091105 
                 SEQ ID NO: 811 pTet/Mnt Hybrid Promoter 
                 ... 
               
               
                   
                   
                 agaactgtaatccctatcagtgatagagat 
               
               
                   
               
               
                 BBa_K091106 
                 SEQ ID NO: 812 LsrA/cI hybrid promoter 
                 ...tgttgatttatctaacaccgtgcgtgttga 
               
               
                   
               
               
                 BBa_K091107 
                 SEQ ID NO: 813 pLux/cI Hybrid Promoter 
                 ... 
               
               
                   
                   
                 acaccgtgcgtgttgatatagtcgaataaa 
               
               
                   
               
               
                 BBa_K091143 
                 SEQ ID NO: 814 pLas/cI Hybrid Promoter 
                 ... 
               
               
                   
                   
                 ggttctttttggtacctctggcggtgataa 
               
               
                   
               
               
                 BBa_K091146 
                 SEQ ID NO: 815 pLas/Lux Hybrid Promoter 
                 ... 
               
               
                   
                   
                 tgtaggatcgtacaggtataaattcttcag 
               
               
                   
               
               
                 BBa_K091157 
                 SEQ ID NO: 816 pLux/Las Hybrid Promoter 
                 ...ctatctcatttgctagtatagtcgaataaa 
               
               
                   
               
               
                 BBa_K094120 
                 SEQ ID NO: 817 pLacI/ara-1 
                 ... 
               
               
                   
                   
                 aattgtgagcggataacaatttcacacaga 
               
               
                   
               
               
                 BBa_K100000 
                 SEQ ID NO: 818 Natural Xylose Regulated Bi- 
                 ...gttacgtttatcgcggtgattgttacttat 
               
               
                   
                 Directional Operator 
               
               
                   
               
               
                 BBa_K101000 
                 SEQ ID NO: 819 Dual-Repressed Promoter for 
                 ... 
               
               
                   
                 p22 mnt and TetR 
                 acggtgacctagatctccgatactgagcac 
               
               
                   
               
               
                 BBa_K101001 
                 SEQ ID NO: 820 Dual-Repressed Promoter for 
                 ... 
               
               
                   
                 LacI and LambdacI 
                 tggaattgtgagcggataaaatttcacaca 
               
               
                   
               
               
                 BBa_K101002 
                 SEQ ID NO: 821 Dual-Repressed Promoter for 
                 ...tagtagataatttaagtgttctttaatttc 
               
               
                   
                 p22 cII and TetR 
               
               
                   
               
               
                 BBa_K109200 
                 SEQ ID NO: 822 AraC and TetR promoter 
                 ... 
               
               
                   
                 (hybrid) 
                 aacaaaaaaacggatcctctagttgcggcc 
               
               
                   
               
               
                 BBa_K112118 
                 SEQ ID NO: 823 rrnB P1 promoter 
                 ... 
               
               
                   
                   
                 ataaatgcttgactctgtagcgggaaggcg 
               
               
                   
               
               
                 BBa_K112318 
                 SEQ ID NO: 824 {&lt;bolA promoter&gt;} in BBb 
                 ... 
               
               
                   
                 format 
                 atttcatgatgatacgtgagcggatagaag 
               
               
                   
               
               
                 BBa_K112322 
                 SEQ ID NO: 825 {Pdps} in BBb format 
                 ... 
               
               
                   
                   
                 gggacacaaacatcaagaggatatgagatt 
               
               
                   
               
               
                 BBa_K112402 
                 SEQ ID NO: 826 promoter for FabA gene— 
                 ... 
               
               
                   
                 Membrane Damage and Ultrasound Sensitive 
                 gtcaaaatgaccgaaacgggtggtaacttc 
               
               
                   
               
               
                 BBa_K112405 
                 SEQ ID NO: 827 Promoter for CadA and CadB 
                 ... 
               
               
                   
                 genes 
                 agtaatcttatcgccagtttggtctggtca 
               
               
                   
               
               
                 BBa_K112406 
                 SEQ ID NO: 828 cadC promoter 
                 ... 
               
               
                   
                   
                 agtaatcttatcgccagtttggtctggtca 
               
               
                   
               
               
                 BBa_K112701 
                 SEQ ID NO: 829 hns promoter 
                 ... 
               
               
                   
                   
                 aattctgaacaacatccgtactcttcgtgc 
               
               
                   
               
               
                 BBa_K116001 
                 SEQ ID NO: 830 nhaA promoter, that can be 
                 ... 
               
               
                   
                 regulated by pH and nhaR protein. 
                 cgatctattcacctgaaagagaaataaaaa 
               
               
                   
               
               
                 BBa_K116500 
                 SEQ ID NO: 831 OmpF promoter that is 
                 ... 
               
               
                   
                 activated or repressed by OmpR according to osmolarity. 
                 aaacgttagtttgaatggaaagatgcctgc 
               
               
                   
               
               
                 BBa_K121011 
                 SEQ ID NO: 832 promoter (lacI regulated) 
                 ... 
               
               
                   
                   
                 acaggaaacagctatgaccatgattacgcc 
               
               
                   
               
               
                 BBa_K136010 
                 SEQ ID NO: 833 fliA promoter 
                 ... 
               
               
                   
                   
                 gttcactctataccgctgaaggtgtaatgg 
               
               
                   
               
               
                 BBa_K145150 
                 SEQ ID NO: 834 Hybrid promoter: HSL-LuxR 
                 ...tagtttataatttaagtgttctttaatttc 
               
               
                   
                 activated, P22 C2 repressed 
               
               
                   
               
               
                 BBa_K145152 
                 SEQ ID NO: 835 Hybrid promoter: P22 c2, 
                 ... 
               
               
                   
                 LacI NOR gate 
                 gaaaatgtgagcgagtaacaacctcacaca 
               
               
                   
               
               
                 BBa_K259005 
                 SEQ ID NO: 836 AraC Rheostat Promoter 
                 ...ttttatcgcaactctctactgtttctccat 
               
               
                   
               
               
                 BBa_K259007 
                 SEQ ID NO: 837 AraC Promoter fused with 
                 ... 
               
               
                   
                 RBS 
                 gtttctccattactagagaaagaggggaca 
               
               
                   
               
               
                 BBa_K266005 
                 SEQ ID NO: 838 PAI + LasR -&gt; LasI &amp; 
                 ... 
               
               
                   
                 AI + LuxR -- LasI 
                 aataactctgatagtgctagtgtagatctc 
               
               
                   
               
               
                 BBa_K266006 
                 SEQ ID NO: 839 PAI + LasR -&gt; LasI + GFP &amp; 
                 ... 
               
               
                   
                 AI + LuxR -- LasI + GFP 
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_K266007 
                 SEQ ID NO: 840 Complex QS -&gt; LuxI &amp; LasI 
                 ... 
               
               
                   
                 circuit 
                 caccttcgggtgggcctttctgcgtttata 
               
               
                   
               
               
                 BBa_R0065 
                 SEQ ID NO: 841 Promoter (lambda cI and luxR 
                 ... 
               
               
                   
                 regulated -- hybrid) 
                 gtgttgactattttacctctggcggtgata 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Examples of Combination Inducible &amp; Repressible 
               
               
                 Miscellaneous Prokaryotic Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_K125100 
                 SEQ ID NO: 842 nir promoter 
                 ... 
               
               
                   
                 from  Synechocystis  sp. PCC6803 
                 cgaaacgggaaccctatattgatctctact 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 Examples of Combination Inducible &amp; Repressible 
               
               
                 Miscellaneous Yeast Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_I766200 
                 SEQ ID NO: 843 pSte2 
                 ... 
               
               
                   
                   
                 accgttaagaaccatatccaagaatcaaaa 
               
               
                   
               
               
                 BBa_K110016 
                 SEQ ID NO: 844 A-Cell Promoter STE2 
                 ... 
               
               
                   
                 (backwards) 
                 accgttaagaaccatatccaagaatcaaaa 
               
               
                   
               
               
                 BBa_K165034 
                 SEQ ID NO: 845 Zif268-HIV bs + LexA bs + 
                 ... 
               
               
                   
                 mCYC promoter 
                 cacaaatacacacactaaattaataactag 
               
               
                   
               
               
                 BBa_K165041 
                 SEQ ID NO: 846 Zif268-HIV binding sites + 
                 ... 
               
               
                   
                 TEF constitutive yeast promoter 
                 atacggtcaacgaactataattaactaaac 
               
               
                   
               
               
                 BBa_K165043 
                 SEQ ID NO: 847 Zif268-HIV binding sites + 
                 ... 
               
               
                   
                 MET25 constitutive yeast promoter 
                 tagatacaattctattacccccatccatac 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Examples of Combination Inducible &amp; Repressible 
               
               
                 Miscellaneous Eukaryotic Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
               
               
                   
               
               
                 BBa_J05215 
                 SEQ ID NO: 848 Regulator for R1- 
                 ... 
               
               
                   
                 CREBH 
                 ggggcgagggccccgcctccggaggcgggg 
               
               
                   
               
               
                 BBa_J05216 
                 SEQ ID NO: 849 Regulator for R3-ATF6 
                 ... 
               
               
                   
                   
                 gaggggacggctccggccccggggccggag 
               
               
                   
               
               
                 BBa_J05217 
                 SEQ ID NO: 850 Regulator for R2- 
                 ... 
               
               
                   
                 YAP7 
                 ggggcgagggctccggccccggggccggag 
               
               
                   
               
               
                 BBa_J05218 
                 SEQ ID NO: 851 Regulator for R4-cMaf 
                 ... 
               
               
                   
                   
                 gaggggacggccccgcctccggaggcgggg 
               
               
                   
               
            
           
         
       
     
     In addition to the above-described promoter sequences, the cassettes and switches described herein can comprise, in addition, one or more molecular species, including, but not limited to, ribosome binding sequences, degradation tag sequences, translational terminator sequences, and anti-sense sequences, that are added to, for example, enhance translation of mRNA sequences for protein synthesis, prevent further transcription downstream of the an encoded protein, or enhance degradation of an mRNA sequence or protein sequence. Such additional molecular species, by enhancing the fidelity and accuracy of the molecular circuits described herein permit, for example, increased numbers and combinations of molecular circuits and improve the capabilities of the molecular circuits described herein. Known enhancer and repressor sequences from promoter regions or intronic regions and their corresponding regulatory proteins or RNAs can also be used to regulate, e.g., transcription. 
     Terminators 
     Provided herein are terminator sequences for use in some embodiments of the invention. A “terminator”, “transcriptional terminator”, “terminator sequence,” as used herein, is a nucleic acid sequence that causes transcription to stop. A terminator may be unidirectional or bidirectional. It is comprised of a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase. A terminator sequence prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters. Thus, in certain embodiments, a terminator that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable output expression levels (e.g., low output levels). 
     The most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid sequence that is usually transcribed, a forward transcriptional terminator will cause transcription to abort. In some embodiments, bidirectional transcriptional terminators are provided, which usually cause transcription to terminate on both the forward and reverse strand. In some embodiments, reverse transcriptional terminators are provided, which usually terminate transcription on the reverse strand only. 
     In prokaryotic systems, terminators usually fall into two categories (1) rho-independent terminators and (2) rho-dependent terminators. Rho-independent terminators are generally composed of palindromic sequence that forms a stem loop rich in G-C base pairs followed by several T bases. Without wishing to be bound by theory, the conventional model of transcriptional termination is that the stem loop causes RNA polymerase to pause, and transcription of the poly-A tail causes the RNA:DNA duplex to unwind and dissociate from RNA polymerase. 
     In eukaryotic systems, the terminator region may comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in some embodiments involving eukaryotes, a terminator may comprise a signal for the cleavage of the RNA. In some embodiments, the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements may serve to enhance output nucleic acid levels and/or to minimize read through between nucleic acids. 
     Terminators for use in accordance with the invention include any terminator of transcription described herein or known to one of ordinary skill in the art. Examples of terminators include, without limitation, the termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences such as, for example, the SV40 terminator, spy, yejM, secG-leuU, thrLABC, rrnB Tl, hisLGDCBHAFI, metZWV, rrnC, xapR, aspA and arcA terminator. In some embodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as those resulting from a sequence truncation. 
     Terminators for use as molecular species in the molecular circuits and modular functional blocks described herein can be selected from the non-limiting examples of Tables 37-41. 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Examples of Forward Terminators 
               
            
           
           
               
               
               
            
               
                   
                 Efficiency 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Name 
                 Description 
                 Direction 
                 Fwd. 
                 Rev. 
                 Length 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 BBa_B0010 
                 T1 from  E. coli  rrnB 
                 Forward 
                   
                   
                 80 
               
               
                 BBa_B0012 
                 TE from coliphageT7 
                 Forward 
                 0.309[CC] 
                 −0.368[CC] 
                 41 
               
               
                 BBa_B0013 
                 TE from coliphage T7 (+/−) 
                 Forward 
                 0.6[CC] 
                 −1.06[CC] 
                 47 
               
               
                 BBa_B0015 
                 double terminator (B0010-B0012) 
                 Forward 
                 0.984[CC] 
                  0.295[CC] 
                 129 
               
               
                   
                   
                   
                 0.97[JK] 
                  0.62[JK] 
               
               
                 BBa_B0017 
                 double terminator (B0010-B0010) 
                 Forward 
                   
                   
                 168 
               
               
                 BBa_B0053 
                 Terminator (His) 
                 Forward 
                   
                   
                 72 
               
               
                 BBa_B0055 
                 -- No description -- 
                   
                   
                   
                 78 
               
               
                 BBa_B1002 
                 Terminator (artificial, small, % T~=85%) 
                 Forward 
                 0.98[CH] 
                   
                 34 
               
               
                 BBa_B1003 
                 Terminator (artificial, small, % T~=80) 
                 Forward 
                 0.83[CH] 
                   
                 34 
               
               
                 BBa_B1004 
                 Terminator (artificial, small, % T~=55) 
                 Forward 
                 0.93[CH] 
                   
                 34 
               
               
                 BBa_B1005 
                 Terminator (artificial, small, % T~=25% 
                 Forward 
                 0.86[CH] 
                   
                 34 
               
               
                 BBa_B1006 
                 Terminator (artificial, large, % T~&gt;90) 
                 Forward 
                 0.99[CH] 
                   
                 39 
               
               
                 BBa_B1010 
                 Terminator (artificial, large, % T~&lt;10) 
                 Forward 
                 0.95[CH] 
                   
                 40 
               
               
                 BBa_I11013 
                 Modification of biobricks part BBa_B0015 
                   
                   
                   
                 129 
               
               
                 BBa_I51003 
                 -- No description -- 
                   
                   
                   
                 110 
               
               
                 BBa_J61048 
                 [rnpB-T1] Terminator 
                 Forward 
                 0.98[JCA] 
                   
                 113 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Examples of Bidirectional Terminators 
               
            
           
           
               
               
               
            
               
                   
                 Efficiency 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Name 
                 Description 
                 Direction 
                 Fwd. 
                 Rev. 
                 Length 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 BBa_B0011 
                 LuxICDABEG (+/−) 
                 Bidirectional 
                 0.419[CC]/ 
                 0.636[CC]/ 
                 46 
               
               
                   
                   
                   
                 0.95[JK] 
                 0.86[JK] 
               
               
                 BBa_B0014 
                 double terminator (B0012-B0011) 
                 Bidirectional 
                 0.604[CC]/ 
                 0.86[JK] 
                 95 
               
               
                   
                   
                   
                 0.96[JK] 
               
               
                 BBa_B0021 
                 LuxICDABEG (+/−), reversed 
                 Bidirectional 
                 0.636[CC]/ 
                 0.419[CC]/ 
                 46 
               
               
                   
                   
                   
                 0.86[JK] 
                 0.95[JK] 
               
               
                 BBa_B0024 
                 double terminator (B0012-B0011), 
                 Bidirectional 
                 0.86[JK] 
                 0.604[CC]/ 
                 95 
               
               
                   
                 reversed 
                   
                   
                 0.96[JK] 
               
               
                 BBa_B0050 
                 Terminator (pBR322, +/−) 
                 Bidirectional 
                   
                   
                 33 
               
               
                 BBa_B0051 
                 Terminator (yciA/tonA, +/−) 
                 Bidirectional 
                   
                   
                 35 
               
               
                 BBa_B1001 
                 Terminator (artificial, small, % T~=90) 
                 Bidirectional 
                 0.81[CH] 
                   
                 34 
               
               
                 BBa_B1007 
                 Terminator (artificial, large, % T~=80) 
                 Bidirectional 
                 0.83[CH] 
                   
                 40 
               
               
                 BBa_B1008 
                 Terminator (artificial, large, % T~=70) 
                 Bidirectional 
                   
                   
                 40 
               
               
                 BBa_B1009 
                 Terminator (artificial, large, % T~=40%) 
                 Bidirectional 
                   
                   
                 40 
               
               
                 BBa_K259006 
                 GFP-Terminator 
                 Bidirectional 
                 0.604[CC]/ 
                 0.86[JK] 
                 823 
               
               
                   
                   
                   
                 0.96[JK] 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 Examples of Reverse Terminators 
               
            
           
           
               
               
               
            
               
                   
                 Efficiency 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Name 
                 Description 
                 Direction 
                 Fwd. 
                 Rev. 
                 Length 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 BBa_B0020 
                 Terminator (Reverse B0010) 
                 Reverse 
                   
                   
                 82 
               
               
                 BBa_B0022 
                 TE from coliphageT7, reversed 
                 Reverse 
                 −0.368[CC] 
                 0.309[CC] 
                 41 
               
               
                 BBa_B0023 
                 TE from coliphage T7, 
                 Reverse 
                 −1.06[CC] 
                 0.6[CC] 
                 47 
               
               
                   
                 reversed 
               
               
                 BBa_B0025 
                 double terminator (B0015), 
                 Reverse 
                  0.295[CC]/ 
                 0.984[CC]/ 
                 129 
               
               
                   
                 reversed 
                   
                  0.62[JK] 
                 0.97[JK] 
               
               
                 BBa_B0052 
                 Terminator (rrnC) 
                 Forward 
                   
                   
                 41 
               
               
                 BBa_B0060 
                 Terminator (Reverse B0050) 
                 Bidirectional 
                   
                   
                 33 
               
               
                 BBa_B0061 
                 Terminator (Reverse B0051) 
                 Bidirectional 
                   
                   
                 35 
               
               
                 BBa_B0063 
                 Terminator (Reverse B0053) 
                 Reverse 
                   
                   
                 72 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Examples of Yeast Terminators 
               
            
           
           
               
               
               
            
               
                   
                 Efficiency 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Name 
                 Description 
                 Direction 
                 Fwd. 
                 Rev. 
                 Length 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 BBa_J63002 
                 ADH1 terminator 
                 Forward 
                 225 
               
               
                   
                 from  S. cerevisiae   
               
               
                 BBa_K110012 
                 STE2 terminator 
                 Forward 
                 123 
               
               
                 BBa_Y1015 
                 CycE1 
                   
                 252 
               
               
                   
               
            
           
         
       
     
                     TABLE 41                  Examples of Eukaryotic Terminators                             Efficiency                                             Name   Description   Direction   Fwd.   Rev.   Chassis   Length               BBa_J52016   eukaryotic - derived from SV40 early poly A signal   Forward               238           sequence       BBa_J63002   ADH1 terminator from  S. cerevisiae     Forward               225       BBa_K110012   STE2 terminator   Forward               123       BBa_Y1015   CycE1                   252                    
Target Genes and Output Products
 
     A variety of target genes and output products are provided for use in accordance with the invention. As used herein, “output products” refer to gene products that may be used as markers of specific states of the logic gates and systems described herein. A target gene of the invention can encode for a protein or RNA that is used to track or mark the state of the cell upon receiving a particular input. Such output products can be used to distinguish between various states (e.g., “ON” or “OFF”) of a cell. Representative output products for the logic cassettes and systems of the invention include, without limitation, reporter proteins, transcriptional repressors, transcriptional activators, selection markers, enzymes, receptor proteins, ligand proteins, RNAs, riboswitches, short-hairpin RNAs and recombinases. Aspects of the invention relate to logic cassettes and systems that include a plurality of logic gates {e.g., at least two logic gates). It should be understood that in such systems, each logic gate may include one or more different output nucleic acid (e.g., that encode(s) different, or unique, output product(s)). Thus, a single cell or system may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different output nucleic acids. 
     In some embodiments, the target gene of the invention may encode a “reporter” or “reporter molecule.” Such target gene is also called the reporter gene. As used herein, a reporter refers to a protein that can be used to measure gene expression and generally produce a measurable signal such as fluorescence, luminescence or color. The presence of a reporter in a cell or organism is readily observed. For example, fluorescent proteins (e.g., GFP, red fluorescent protein such as mCherry) cause a cell to fluoresce when excited with light of a particular wavelength, luciferases cause a cell to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate to a colored product. In some embodiments, reporters may be used to quantify the strength or activity of the input received by the systems of the invention. In some embodiments, reporters can be fused in-frame to other protein coding sequences to identify where a protein is located in a cell or organism. Reporters for use in accordance with the invention include any reporter described herein or known to one of ordinary skill in the art. 
     There are several different ways to measure or quantify a reporter depending on the particular reporter and what kind of characterization data is desired. In some embodiments, microscopy can be a useful technique for obtaining both spatial and temporal information on reporter activity, particularly at the single cell level. In some embodiments, flow cytometers can be used for measuring the distribution in reporter activity across a large population of cells. In some embodiments, plate readers may be used for taking population average measurements of many different samples over time. In some embodiments, instruments that combine such various functions, may be used, such as multiplex plate readers designed for flow cytometers, and combination microscopy and flow cytometric instruments. 
     Fluorescent proteins may be used for visualizing or quantifying the output of logic gates/systems. Fluorescence can be readily quantified using a microscope, plate reader or flow cytometer equipped to excite the fluorescent protein with the appropriate wavelength of light. Several different fluorescent proteins are available, thus multiple gene expression measurements can be made in parallel. Examples of genes encoding fluorescent proteins that may be used in accordance with the invention include, without limitation, those proteins provided in U.S. Patent Application No. 2012/0003630 (see Table 59), incorporated herein by reference. 
     Examples of UV fluorescent proteins include, but are not limited to, Sirius. Examples of blue fluorescent proteins include, but are not limited to, Azurite, EBFP2, mKalama1, mTagBFP2, and tagBFP. Examples of cyan fluorescent proteins include, but are not limited to, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, Mtfp1, monomeric Midoriishi-Cyan, and Aquamarine. Examples of green fluorescent proteins include, but are not limited to, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, and mNeonGreen. Examples of yellow fluorescent proteins include, but are not limited to, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, IanRFP-Δ583, and mPapayal. Examples of orange fluorescent proteins include, but are not limited to, Monomeric Kusabira-Orange, mOrange, mOrange2, mKOκ, and Mko2. Examples of red fluorescent proteins include, but are not limited to, TagRFP, TagRFP-T, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mCherry, and mNectarine. Examples of far red fluorescent proteins include, but are not limited to, mKate2, HcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, and mCardinal. Examples of near IR fluorescent proteins include, but are not limited to, iFP1.4, iRFP713 (iRFP), iRFP670, iRFP682, iRFP702, iRFP720, and iFP2.0. Examples of sapphire-type fluorescent proteins include, but are not limited to, Sapphire, T-Sapphire, and mAmetrine. Examples of long Stokes shift fluorescent proteins include, but are not limited to, mKeima Red, mBeRFP, LSS-mKate2, LSS-mKate1, and LSSmOrange. 
     Luciferases may also be used for visualizing or quantifying the output of logic gates/systems, particularly for measuring low levels of gene expression, as cells tend to have little to no background luminescence in the absence of a luciferase Luminescence can be readily quantified using a plate reader or luminescence counter. Examples of genes encoding luciferases for that may be used in accordance with the invention include, without limitation, dmMyD88-linker-Rluc, dmMyD88-linker-Rluc-linker-PEST191,  Renilla luciferase , and firefly luciferase (from Photinus pyralis). 
     Enzymes that produce colored substrates (“colorimetric enzymes”) may also be used for visualizing or quantifying the output of logic gates/systems. Enzymatic products may be quantified using spectrophotometers or other instruments that can take absorbance measurements including plate readers. Like luciferases, enzymes such as β-galactosidase can be used for measuring low levels of gene expression because they tend to amplify low signals. Examples of genes encoding colorimetric enzymes that may be used in accordance with the invention include, without limitation, lacZ alpha fragment, lacZ (encoding beta-galactosidase, full-length), and xylE. 
     In some embodiments, the reporter molecule is chloramphenicol acetyltransferase. In some embodiments, the reporter molecule is neomycin phosphotransferase. In some embodiments, the reporter molecule is Secreted Placental Alkaline Phosphatase (SEAP). In some embodiments, the reporter molecule is secreted α-amylase (SAMY). 
     Transcriptional Outputs 
     In some embodiments, the target gene of the invention may encode a transcriptional activator or repressor, the production of which by an output gene can result in a further change in state of the cell, and provide additional input signals to subsequent or additional logic gates. Transcriptional regulators either activate or repress transcription from cognate promoters. Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase. Other transcriptional regulators serve as either an activator or a repressor depending on where it binds and cellular conditions. Transcriptional regulators for use in accordance with the invention include any transcriptional regulator described herein or known to one of ordinary skill in the art. Examples of genes encoding transcriptional regulators that may be used in accordance with the invention include, without limitation, those regulators provided in U.S. Patent Application No. 2012/0003630, incorporated herein by reference. 
     Selection Marker Outputs 
     In some embodiments, the target gene of the invention may encode a selection marker. As used herein, a “selection marker” refers to protein coding sequence that confers a selective advantage or disadvantage to a biological unit, such as a cell. For example, a common type of prokaryotic selection marker is one that confers resistance to a particular antibiotic. Thus, cells that carry the selection marker can grow in media despite the presence of antibiotic. For example, most plasmids contain antibiotic selection markers so that it is ensured that the plasmid is maintained during cell replication and division, as cells that lose a copy of the plasmid will soon either die or fail to grow in media supplemented with antibiotic. A second common type of selection marker, often termed a positive selection marker, is toxic to the cell. Positive selection markers are frequently used during cloning to select against cells transformed with the cloning vector and ensure that only cells transformed with a plasmid containing the insert. Selection markers for use in accordance with the invention include any selection marker described herein or known to one of ordinary skill in the art. Examples of genes encoding selection markers that may be used in accordance with the invention include, without limitation, those markers provided in U.S. Patent Application No. 2012/0003630, incorporated herein in its entirety by reference. 
     Enzyme Outputs 
     In some embodiments, the target gene of the invention may encode an enzyme. In some embodiments, an enzyme is used as a response to a particular input. For example, in response to a particular input received by a logic and memory system of the invention, such as a certain range of toxin concentration present in the environment, the system may turn “ON” a logic gate containing a target gene that encodes an enzyme that can degrade or otherwise destroy the toxin. 
     In some embodiments, output products may be “biosynthetic enzymes” that catalyze the conversion of substrates to products. For example, such biosynthetic enzymes can be used in accordance with the invention to assemble pathways that produce or degrade useful chemicals and materials, in response to specific signals. These combinations of enzymes can reconstitute either natural or synthetic biosynthetic pathways. These enzymes have applications in specialty chemicals, biofuels and bioremediation. Enzymes for use in accordance with the invention include any enzyme described herein or known to one of ordinary skill in the art. Examples of genes encoding enzymes that may be used in accordance with the invention include, without limitation, those provided in U.S. Patent Application No. 2012/0003630, incorporated herein by reference. 
     Receptors, Ligands and Lytic Proteins 
     In some embodiments, the target gene of the invention may encode a receptor, ligand or lytic protein. Receptors tend to have three domains: an extracellular domain for binding ligands such as proteins, peptides or small molecules, a transmembrane domain and an intracellular or cytoplasmic domain, which frequently can participate in some sort of signal transduction event such as phosphorylation. In some embodiments, transporters, channels or pumps are used as output products. Transporters are membrane proteins responsible for transport of substances across the cell membrane. Channels are made up of proteins that form transmembrane pores through which selected ions can diffuse. 
     Pumps are membrane proteins that can move substances against their gradients in an energy-dependent process known as active transport. In some embodiments, nucleic acid sequences encoding proteins and protein domains whose primary purpose is to bind other proteins, ions, small molecules, and other ligands may be used in accordance with the invention. Receptors, ligands and lytic proteins for use in accordance with the invention include any receptor, ligand and lytic protein, described herein or known to one of ordinary skill in the art. 
     Examples of genes encoding receptors, ligands and lytic proteins that may be used in accordance with the invention include, without limitation, those provided in U.S. Patent Application No. 2012/0003630, incorporated herein by reference. 
     A target gene can be any gene of interest, e.g., a gene that encodes a therapeutic protein or peptide of interest, a therapeutic protein inhibitor, miRNA, siRNA, etc., nucleic acid inhibitor (e.g., RNAi) etc., antibody or fragment thereof. Exemplary therapeutic proteins include, but are not limited to, antibodies or fragments thereof, CAR for cancer therapy as disclosed herein. 
     In some embodiments, the target gene of the invention may encode a RNA molecule of interest. For example, the RNA molecule of interest can be an sgRNA from the CRISPR/Cas9 system. 
     Genetic Engineering of Logic Gates and Systems 
     A cell to be engineered for use with the logic cassettes, switches, and systems of the invention may be any cell or host cell. As defined herein, a “cell” or “cellular system” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular. The logic cassettes, switches, and systems described herein can be used in a cell of a variety of organisms. Preferably, the host cell is a mammalian cell. More preferably, the host cell is a human cell. The logic cassettes, switches, and systems described herein can be used in a variety of cell types. Exemplary cell types include, but are not limited to, liver cells, gastrointestinal cells, epithelial cells, endothelial cells, kidney cells, cancer cells, blood cells, stem cells, bone cells, smooth muscle cells, striated muscle cells, cardiac muscle cells, immune cells and nerve cells. Blood cells include, e.g., leukocytes, such as neutrophils, lymphocytes, monocytes, eosinophils, basophils, macrophages Immune cells include, but are not limited to, monocytes, Natural Killer (NK) cells, dendritic cells (which could be immature or mature), subsets of dendritic cells including myeloid, plasmacytoid (also called lymphoid) or Langerhans; macrophages such as histiocytes, Kupffer&#39;s cells, alveolar macrophages or peritoneal macrophages; neutrophils, eosinophils, mast cells, basophils; B cells including plasma B cells, memory B cells, B-1 cells, B-2 cells; CD45RO (naive T), CD45RA (memory T); CD4 Helper T Cells including Th1, Th2 and Tr1/Th3; CD8 Cytotoxic T Cells, Regulatory T Cells and Gamma Delta T Cells. 
     In some embodiments, a cell for use in accordance with the invention is an eukaryotic cell, which comprises membrane-bound compartments in which specific metabolic activities take place, such as a nucleus. Examples of eukaryotic cells for use in accordance with the invention include, without limitation, mammalian cells, insect cells, yeast cells {e.g.,  Saccharomyces cerevisiae ) and plant cells. In some embodiments, the eukaryotic cells are from a vertebrate animal. Examples of vertebrate cells for use in accordance with the invention include, without limitation, reproductive cells including sperm, ova and embryonic cells, and non-reproductive cells, including kidney, lung, spleen, lymphoid, cardiac, gastric, intestinal, pancreatic, muscle, bone, neural, brain and epithelial cells. Stem cells, including embryonic stem cells, can also be used. 
     In some embodiments, a cell for use in accordance with the invention is a prokaryotic cell, which may comprise a cell envelope and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. In some embodiments, the cells are bacterial cells. As used herein, the term “bacteria” encompasses all variants of bacteria, for example, prokaryotic organisms and cyanobacteria. Bacteria are small (typical linear dimensions of around 1 micron), non-compartmentalized, with circular DNA and ribosomes of 70S. The term “bacteria” also includes bacterial subdivisions of Eubacteria and Archaebacteria. Eubacteria can be further subdivided into gram-positive and gram-negative Eubacteria, which depend upon a difference in cell wall structure. Also included herein are those classified based on gross morphology alone (e.g., cocci, bacilli). In some embodiments, the bacterial cells are gram-negative cells, and in some embodiments, the bacterial cells are gram-positive cells. Examples of bacterial cells that may be used in accordance with the invention include, without limitation, cells from  Yersinia  spp.,  Escherichia  spp.,  Klebsiella  spp.,  Bordetella  spp.,  Neisseria  spp.,  Aeromonas  spp.,  Franciesella  spp.,  Corynebacterium  spp.,  Citrobacter  spp.,  Chlamydia  spp.,  Hemophilus  spp.,  Brucella  spp.,  Mycobacterium  spp.,  Legionella  spp.,  Rhodococcus  spp.,  Pseudomonas  spp.,  Helicobacter  spp.,  Salmonella  spp.,  Vibrio  spp.,  Bacillus  spp.,  Erysipelothrix  spp.,  Salmonella  spp.,  Stremtomyces  spp. In some embodiments, the bacterial cells are from  Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Streptomyces, Actinobacillus actinobycetemcomitans, Bacteroides, cyanobacteria, Escherichia coli, Helobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus planta rum, Streptococcus faecalis, Bacillus coagulans, Bacillus ceretus, Bacillus popillae, Synechocystis  strain PCC6803,  Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferns, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromo  genes,  Streptomyces ghanaenis, Halobacterium  strain GRB, or  Halobaferax  sp. strain Aa2.2. 
     In some embodiments, a non-cellular system such as a virus or phage may be used in accordance with the invention. For examples, any one or more component(s) of the synthetic logic and memory systems may be introduced, by direct integration of logic system nucleic acids, for example, into a viral genome. A virus for use as described herein may be a double-stranded DNA (dsDNA) virus (e.g., Adenoviruses, Herpesviruses, Poxviruses), a single-stranded DNA (ssDNA) viruses ((+)sense DNA) (e.g. Parvoviruses); a double-stranded RNA (dsRNA) virus (e.g., Reoviruses); a (+)ssRNA viruses ((+)sense RNA) (e.g. Picornaviruses, Togaviruses); (−)ssRNA virus ((−)sense RNA) (e.g., Orthomyxoviruses, Rhabdo viruses); a single-stranded RNA (ssRNA)-Reverse Transcriptase viruses ((+)sense RNA with DNA intermediate in life-cycle) (e.g., Retroviruses); or a dsDNA-Reverse Transcriptase virus (e.g., Hepadnaviruses). 
     Viruses may also include plant viruses and bacteriophages or phages. Examples of phage families that may be used in accordance with the invention include, without limitation, Myoviridae (T4-like viruses; P1-like viruses; P2-like viruses; Mu-like viruses; SPO1-like viruses; phiH-like viruses); Siphoviridae γ-like viruses (T1-like viruses; T5-like viruses; c2-like viruses; L5-like viruses; .psi.M1-like viruses; phiC31-like viruses; N15-like viruses); Podoviridae (T7-like viruses; phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae ( Tecti  virus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudi virus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectro virus); Microviridae (Micro virus, Spiromicro virus, Bdellomicro virus, Chlamydiamicro virus); Leviviridae (Levi virus, Allolevivirus) and Cystoviridae (Cysto virus). Such phages may be naturally occurring or engineered phages. 
     In some embodiments, the cell or cellular system is a “natural cell” (e.g., found in nature; not artificial or synthetic). In some embodiments, the cell or cellular system is an artificial cell or synthetic cell. As used herein, an “artificial cell” or a “synthetic cell” is a minimal cell formed from artificial parts that can function in ways that a natural cell can function (e.g., transcribe and translate proteins and generate ATP). 
     A host cell in accordance with the invention includes any host cell that, upon transformation or transfection with one or more component(s) of the synthetic logic system (e.g., logic gates) is capable of supporting the activation and expression of the synthetic logic and memory system component(s). 
     In some embodiments, one or more component(s) of the synthetic logic and memory systems of the invention may be introduced into a cellular or non-cellular system using a vector or plasmid. As used herein, a “vector” is used interchangeably with “plasmid” to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors capable of directing the expression of genes and/or nucleic acid sequence to which they are operatively linked are referred to herein as “expression vectors.” In general, expression vectors described herein are often in the form of plasmids, which are circular double-stranded DNA loops not bound to chromosome. Expression vectors may be vectors for stable or transient expression of the DNA. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome. Other expression vectors may be used in accordance with the invention including, without limitation, episomes, bacteriophages and viral vectors, and such vectors can integrate into the host&#39;s genome or replicate autonomously in the particular cellular system used. Other forms of expression vectors known by those skilled in the art which serve the equivalent functions may also be used. 
     Vectors comprising nucleic acid sequences of the invention (e.g., those encoding logic gates) may be “introduced” into cells as polynucleotides by techniques well-known in the art for introducing DNA and RNA into cells. As used herein, “transfection” refers to the introduction of genetic material (e.g., a vector comprising nucleic acid sequences) into a cell, tissue or organism. Transfection of a cell may be stable or transient. A host cell is considered to be transiently transfected when nucleic acid is introduced into the cell and does not integrate into the host cell&#39;s genome. Transient transfection may be detected by, for example, enzyme linked immunosorbent assay (ELISA), which detects the presence of a polypeptide encoded by the nucleic acid, or it may be detected by detecting the activity of a protein encoded by the nucleic acid. By contrast, a host cell is considered to be stably transfected when nucleic acid is introduced into the cell and does integrate into the host cell&#39;s genome. Stably transfected cells pass the introduced nucleic acid to their progeny (i.e., stable heritability through meiosis). Stable transfection of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes, or by polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences. 
     In some embodiments, a synthetic RNA-processing platform may be used to process mRNAs to separate the 5′ untranslated region (UTR) from the downstream gene. For example, a sequence encoding a recombinase as disclosed herein may be inserted upstream (e.g., directly upstream) of the output nucleic acid to improve expression of the gene. In some embodiments, the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) pathway may be used to process mRNAs to separate the 5′ UTR from the downstream gene. In some embodiments, a coupled translational system with an upstream “throwaway” open reading frame (ORF) and downstream ORF may be used to generate mRNAs with more reliable and programmable translation of the downstream. Other synthetic RNA-processing platforms are also contemplated herein. 
     Uses of Synthetic Cassettes, Switches, and Systems 
     The components of the nucleic acid logic cassettes or switches shown are exemplary. One skilled in the art can readily substitute the different genetic elements such as promoters and RRS in the cassettes or switches. For example, one can readily substitute the recombinases and integrases depending on the RRS and/or the cell into which the cassette is to be inserted. The different examples of genetic elements such as promoters, RRS, and recombinases as discussed can be used in any combinations to construct a cassette or switch capable of a desirable logic function. The target genes used can readily be selected based upon the cell and desired use. For example, the target gene used in the cassettes or switches can be a regulatory gene, a marker gene, or a therapeutic gene. 
     The nucleic acid logic cassettes and switches of the invention can be constructed using methods known in the art. In some embodiments, a Gibson isothermal assembly method can be used. 
     The nucleic acid logic cassettes, switches, and systems of the invention are useful for, inter alia, engineering complex behavioral phenotypes in cellular systems, such as prokaryotic, eukaryotic and synthetic cells, or in non-cellular systems, including cell-free systems, test tubes, viruses and phages. The logic cassettes and systems described herein combine the power of nucleic acid-based engineering methods with computational and systems biology approaches for programming cellular, or biological, state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications. As used herein, a “state machine” refers to any tool that stores the status (or state) of something at a given time and can operate on input(s) to change the status and/or cause an action(s) or output(s) to take place for any given change. Typically, a state machine includes a set of input events, a set of output events, a set of states, a function that maps states and input(s) to output(s), a function that maps states and inputs to new states (which is referred to as a state transition function), and a description of the initial state. 
     The synthetic logic cassettes, switches, systems of the invention may be used for a variety of applications and in many different types of methods, including, but not limited to, bioremediation, biosensing and biomedical therapeutics. In some embodiments, the logic and memory systems may be used to build multiplexed cellular switches for gene expression or synthetic differentiation cascades. Cellular signals can be integrated as inputs to the logic and memory systems by linking the signals to recombinase expression. Multicellular systems endowed with the synthetic logic cassettes, switches, systems of the invention may also implement distributed computation or synthetic cellular consortia. 
     The nucleic acid logic cassettes, switches, and systems of the invention are useful to control gene expression for cell-based immunotherapy (e.g., adoptive T-cell therapy). Exemplary target genes of interest include, but are not limited to, chimeric antigen receptor, T cell receptor, cytokines (e.g., IL-2, IL-12, IL-15), and suicide genes (e.g., HSV-TK, iCasp9). 
     In some embodiments, the synthetic logic cassettes, switches, and systems of the invention may also be used to build “digital-to-analog converters,” which translate digital representations back into analog outputs. Such systems may be used to reliably set internal system states. For example, instead of fine-tuning transcriptional activity with varying amounts of chemical inducers, a digital-to-analog converter, composed of a bank of genetic switches (different recombinases and logic gates), each of which is sensitive to a different inducer, provides better control. By enabling, through each activated switch, transcription from promoters of varying strengths (e.g., P output,3&gt;P output,2&gt;P output,1), digital combinations of inducers may be used to program defined levels of transcriptional activities. Such a circuit may be used in biotechnology applications, where reliable expression of different pathways is needed for programming different modes of operation in engineered cells. In addition, digital-to-analog converters are useful for providing a multiplexed method for probing synthetic circuits. For example, because each analog level is associated with a distinct digital state, a single analog output can allow one to infer the internal digital state of a synthetic gene network. 
     Further, in some embodiments, the synthetic logic cassettes, switches, and systems of the invention may be used for detection of arsenic in drinking water and/or a range of toxins and/or heavy metals. The systems may be coupled to genetically engineered bacteria, which are capable of digesting and neutralizing toxins and heavy metals. This may be achieved, for example, by the bacteria sensing a specific toxin or heavy metal, and the sensor being directly linked as input for an inducible promoter that controls recombinase expression, which in turn activates the logic/memory system by flipping (e.g., activating or de-activating) the gene, promoter or terminator. As a result, the pathway that controls digesting and neutralizing toxins and heavy metals is turned on. 
     The methods and uses of the synthetic logic cassettes, switches, and systems of the invention may involve in vivo, ex vivo, or in vitro systems. The term “in vivo” refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some embodiments, a method or use can be said to occur in vivo when a unicellular organism, such as a bacteria, is used. The term “ex vivo” refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant (e.g., explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissue or cells, including blood cells, among others). The term “in vitro” refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and may refer to introducing an engineered genetic counter in a non-cellular system, such as a media not comprising cells or cellular systems, such as cellular extracts. 
     It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., disclosed herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. 
     As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” 
     Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are disclosed herein. 
     Some embodiments of the invention are listed in the following numbered paragraphs:
         1. A nucleic acid logic cassette comprising:
           (i) a nucleic acid sequence encoding a mammalian promoter;   (ii) a first recombination unit (U1) comprising a first pair of recombinase recognition sequences (RRS1) for a first recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter,
               whereby when the R1 recognizes the RRS1, the first nucleic acid sequence is excised when the RRS1 are in the same orientation or is inverted when the RRS1 are in the inverse orientation;   
               (iii) a second recombination unit (U2) comprising a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1,
               whereby when the R2 recognizes the RRS2, the second nucleic acid sequence is excised when the RRS2 are in the same orientation or is inverted when the RRS2 are in the inverse orientation;   
               (iv) a third recombination unit (U3) comprising a third pair of recombinase recognition sequences (RRS3) adapted to be recognized by the R1, R2, or a third recombinase (R3), wherein each of the RRS3 flanks each side of a third nucleic acid sequence, wherein the RRS3 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS3 is positioned downstream of at least one of the RRS2,
               whereby when the R1, R2, or R3 recognizes the RRS3, the third nucleic acid sequence is excised when the RRS3 are in the same orientation, or is inverted when the RRS3 are in the inverse orientation;   
               wherein the first, second or third nucleic acid sequence comprises a target gene, and wherein presence or absence of at least one of the R1, R2, and R3 operatively links the promoter to at least one of the first, second or third nucleic acid sequence, thereby driving expression of the first, second or third nucleic acid sequence; and   (v) a pair of flanking nucleic acid sequences, wherein each of the flanking nucleic acid sequences flanks each side of the nucleic acid logic cassette, permitting the cassette to be inserted into a mammalian cell.   
           2. The nucleic acid logic cassette of paragraph 1, wherein the mammalian cell is a human cell.   3. The nucleic acid logic cassette of paragraph 1 or 2, wherein the R2 does not recognize the RRS1 or RRS3.   4. The nucleic acid logic cassette of any one of the above paragraphs, wherein the R3 does not recognize the RRS1 or RRS2.   5. The nucleic acid logic cassette of any one of the above paragraphs, adapted to function as a decoder, wherein the decoder can receive n input signals and produce 2 n  output signals, wherein n is an integer greater than 1.   6. The nucleic acid logic cassette of paragraph 5, wherein the decoder is a 2-input 4-output decoder.   7. The nucleic acid logic cassette of paragraph 6, wherein the 2-input 4-output decoder comprises a first target gene (TG1), a second target gene (TG2), a third target gene (TG3), and a fourth target gene (TG4) connected in series, wherein the TG1 is operatively linked to the promoter, whereby (a) when both the R1 and R2 are absent, the TG1 is expressed to produce a first output signal; (b) when the R1 is present and the R2 is absent, the TG2 is operatively linked to the promoter, thereby expressing the TG2 to produce a second output signal; (c) when the R1 is absent and the R2 is present, the TG3 is operatively linked to the promoter, thereby expressing the TG3 to produce a third output signal; and (d) when both the R1 and R2 are present, the TG4 is operatively linked to the promoter, thereby expressing the TG4 to produce a fourth output signal.   8. The nucleic acid logic cassette of paragraph 6 or 7, wherein the RRS2 are in the same orientation with respect to each other and the second nucleic acid sequence comprises the U1 and a first target gene (TG1), wherein the U1 is upstream of the TG1, wherein the TG1 is in the same orientation as the RRS2, and wherein in U1, the RRS1 are in same orientation with respect to each other and the first nucleic acid sequence encodes a second target gene (TG2), wherein the TG2 is in the same orientation as the RRS1; and
           wherein the U3 is located downstream of the U2, and the RRS3 are in the same orientation with respect to each other and the third nucleic acid sequence encodes a third target gene (TG3),   the cassette optionally comprising a fourth target gene (TG4) positioned downstream of the U3, wherein the TG4 is in the same orientation with respect to the RRS3, and   wherein the RRS1 and RRS3 are recognized by the R1, and whereby   
           (a) absence of the R1 and R2 operatively links the promoter to the TG2, or   (b) presence of the R1 and absence of the R2 operatively link the promoter to the TG1, or   (c) absence of the R1 and presence of the R2 operatively link the promoter to the TG3, or   (d) presence of the R1 and R2 operatively links the promoter to the TG4.   9. The nucleic acid logic cassette of paragraph 6 or 7, wherein the U2 has the RRS2 in an inverse orientation with respect to each other and the second nucleic acid sequence comprises the U1 and the U3, wherein the U3 is positioned downstream of the U1, and
           wherein in the U1, the RRS1 are in an inverse orientation with respect to each other and the first nucleic acid sequence comprises a first target gene (TG1) and a second target gene (TG2), wherein the TG1 and the TG2 are in an inverse orientation with respect to each other, and the TG1 is in the same orientation as the promoter, and   wherein in the U3, the RRS3 are in an inverse orientation with respect to each other and the third nucleic acid sequence comprises a third target gene (TG3) and a fourth target gene (TG4), wherein the TG3 and the TG4 are in an inverse orientation with respect to each other, and the TG3 is in the same orientation as the promoter, and   wherein the RRS1 and RRS3 are recognized by the R1, and whereby   
           (a) absence of the R1 and R2 operatively links the promoter to the TG1, or   (b) presence of the R1 and absence of the R2 operatively link the promoter to the TG3, or   (c) absence of the R1 and presence of the R2 operatively link the promoter to the TG4, or   (d) presence of R1 and R2 operatively links the promoter to the TG2.   10. The nucleic acid logic cassette of paragraph 6 or 7, wherein the U2 has the RRS2 in same orientation with respect to each other and the second nucleic acid sequence comprises the U1 upstream of a first target gene (TG1), wherein the TG1 is in the same orientation as the RRS2, and wherein in U1, each of the RRS1 are in same orientation with respect to each other and the first nucleic acid sequence comprises a transcriptional terminator sequence (TT), wherein the TT is in the same orientation as each of the RRS1; and
           wherein the U3 is located downstream of the U2, and the RRS3 are in the same orientation with respect to each other and the third nucleic acid sequence comprises a second target gene (TG2),   the cassette optionally comprising a fourth nucleic acid sequence comprising a third target gene (TG3) located downstream of the U3, wherein the TG3 is in the same orientation with respect to the RRS3, and   wherein the RRS1 and RRS3 are recognized by the R1, and whereby   
           (a) absence of the R1 and R2, none of the TG1, TG2 or TG3 are operatively linked to the promoter, or   (b) presence of the R1 and absence of the R2 operatively link the promoter to the TG1, or   (c) absence of the R1 and presence of the R2 operatively link the promoter to the TG3, or   (d) presence of the R1 and R2 operatively links the promoter to the TG2.   11. The nucleic acid logic cassette of paragraph 6, wherein the 2-input 4-output decoder comprises a transcription terminator, a first target gene, a second target gene, and a third target gene connected in series, wherein the transcription terminator is operatively linked to the promoter, whereby (a) when both the R1 and R2 are absent, no output signal is produced; (b) when the R1 is present and the R2 is absent, the first target gene is operatively linked to the promoter, thereby expressing the first target gene to produce a first output signal; (c) when the R1 is absent and the R2 is present, the second target gene is operatively linked to the promoter, thereby expressing the second target gene to produce a second output signal; and (d) when both the R1 and R2 are present, the third target gene is operatively linked to the promoter, thereby expressing the third target gene to produce a third output signal.   12. The nucleic acid logic cassette of paragraph 5, wherein the decoder is a 3-input 8-output decoder.   13. The nucleic acid logic cassette of paragraph 12, further comprising (i) a first 2-input 4-output decoder of paragraph 8; (ii) a second 2-input 4-output decoder of paragraph 8 positioned downstream of the first 2-input 4-output decoder and in the same orientation as the first 2-input 4-output decoder, (ii) a fourth recombination unit (U4) comprising a fourth pair of recombinase recognition sequences (RRS4) specific for the R3, wherein each of the RRS4 flanks each side of the first 2-input 4-output decoder, and (iii) a target gene positioned downstream of the second 2-input 4-output decoder, wherein the RRS4 are in the same orientation with respect to each other, whereby when R3 recognizes the RRS4, the first nucleic acid logic construct of the 2-input 4-output decoder is excised.   14. The nucleic acid logic cassette of paragraph 12, further comprising (i) a nucleic acid sequence comprising a first 2-input 4-output decoder of paragraph 9 and a second 2-input 4-output decoder of paragraph 9 connected together and in the same orientation, and (ii) a fourth recombination unit (U4) comprising a fourth pair of recombinase recognition sequence (RRS4) specific for a third recombinase (R3), wherein each of the RRS4 flanks each side of the nucleic acid sequence of (i), and wherein the RRS4 are in an inverse orientation with respect to each other, whereby when R3 recognizes the RRS4, the nucleic acid sequence of (i) inverted with respect to the promoter.   15. The nucleic acid logic cassette of any one of paragraphs 1-4, adapted to function as a multi-input AND gate configured to receive at least three inputs, wherein the U1, U2 and U3 are connected in series, wherein:
           in U1, the RRS1 are in an inverse orientation with respect to each other and the first nucleic acid encodes the promoter in an inverted orientation;   in U2, the RRS2 are in the same orientation with respect to each other and the second nucleic acid comprises a transcriptional terminator sequence (TT);   in U3, the RRS3 are in an inverse orientation with respect to each other and the third nucleic acid comprises a target gene (TG) in an inverted orientation;   wherein the RRS1, RRS2, and RRS3 are each recognized by a different recombinase, whereby only in the presence of the R1, R2 and R3 is the TG operatively linked to the promoter.   
           16. The nucleic acid logic cassette of paragraph 15, further comprising a fourth recombination unit (U4) comprising a fourth pair of recombinase recognition sequences (RRS4) for a fourth recombinase (R4) different from the R1, R2, and R3, the RRS4 being in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a fourth nucleic acid sequence comprising a second transcriptional terminator sequence (TT2), wherein the U4 is positioned in between the U2 and U3, whereby only in the presence of the R1, R2, R3, and R4 is the TG operatively linked to the promoter.   17. The nucleic acid logic cassette of any one of paragraphs 1-4, adapted to function as a full adder.   18. The nucleic acid logic cassette of paragraph 17, wherein the U1 has the RRS1 in the same orientation with respect to each other and the first nucleic acid sequence comprises the U2, a fourth recombination unit (U4) positioned downstream of the U2, and a first target gene (TG1) positioned downstream of the U4,
           wherein the U2 has the RRS2 in the same orientation with respect to each other and the second nucleic acid comprises the U3 and a second target gene (TG2) positioned downstream of the U3, wherein the U3 has the RRS3 in the same orientation with respect to each other and the third nucleic acid comprises a transcriptional terminator sequence, wherein the U4 comprises a fourth pair of recombinase recognition sequences (RRS4) in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a second TG2,   further comprising (i) a fifth recombination unit (U5) positioned downstream of the U1 and comprising a fifth pair of recombinase recognition sequences (RRS5) in the same orientation with respect to each other and a fifth nucleic acid sequence, each of the RRS5 flanking each side of the fifth nucleic acid sequence, the fifth nucleic acid sequence comprising a sixth recombination unit (U6) and a second TG1 positioned downstream of the U6, the U6 comprising a sixth pair of recombinase recognition sequences (RRS6) in the same orientation with respect to each other, wherein each of the RRS6 flanks each side of a third TG2; (ii) a seventh recombination unit (U7) positioned downstream of the U5, the U7 comprising a seventh pair of recombinase recognition sequences (RRS7) in the same orientation with respect to each other, wherein each of the RRS7 flanks each side of a third TG1; (iii) a fourth TG2 connected to a fourth TG1 and positioned downstream of the U7,   and wherein the R1 recognizes the RRS1, the R2 recognizes the RRS2, RRS6, and RRS7, and the R3 recognized the RRS3, RRS4, and RRS5.   
           19. The nucleic acid logic cassette of any one of paragraphs 1-4, adapted to function as a full subtractor.   20. The nucleic acid logic cassette of paragraph 19, wherein the U1 has the RRS1 in the same orientation with respect to each other and the first nucleic acid sequence comprises the U2, a fourth recombination unit (U4) positioned downstream of the U2, and a first transcriptional terminator sequence (TT1) downstream of the U4,
           wherein the U2 has the RRS2 in the same orientation with respect to each other and the second nucleic acid comprises the U3 and a first target gene (TG1) positioned downstream of the U3, wherein the U3 has the RRS3 in the same orientation with respect to each other and the third nucleic acid comprises a second transcriptional terminator sequence (TT2), wherein the U4 comprises a fourth pair of recombinase recognition sequences (RRS4) in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a second TG1 and a first TG2 connected in series,   further comprising (i) a fifth recombination unit (U5) positioned downstream of the U1 and comprising a fifth pair of recombinase recognition sequences (RRS5) in the same orientation with respect to each other and a fifth nucleic acid sequence, each of the RRS5 flanking each side of the fifth nucleic acid sequence, the fifth nucleic acid sequence comprising a sixth recombination unit (U6) and a second TG2 positioned downstream of the U6, the U6 comprising a sixth pair of recombinase recognition sequences (RRS6) in the same orientation with respect to each other, wherein each of the RRS6 flanks each side of a third TG1 and a third TG2 connected in series; (ii) a seventh recombination unit (U7) positioned downstream of the U5, the U7 comprising a seventh pair of recombinase recognition sequences (RRS7) in the same orientation with respect to each other, wherein each of the RRS7 flanks each side of a third transcriptional terminator sequence (TT3); (iii) a fourth TG2 connected to a fourth TG1 and positioned downstream of the U7,   and wherein the R1 recognizes the RRS1, the R2 recognizes the RRS2, RRS6, and RRS7, and the R3 recognizes the RRS3, RRS4, and RRS5.   
           21. The nucleic acid logic cassette of any one of paragraphs 1-4, adapted to function as a half adder-subtractor.   22. The nucleic acid logic cassette of paragraph 21, wherein the U1 has the RRS1 in the same orientation with respect to each other and the first nucleic acid sequence comprises the U2, a fourth recombination unit (U4) positioned downstream of the U2, and a first target gene (TG1) positioned downstream of the U4,
           wherein the U2 has the RRS2 in the same orientation with respect to each other and the second nucleic acid comprises the U3 and a second target gene (TG2) positioned downstream of the U3, wherein the U3 has the RRS3 in the same orientation with respect to each other and the third nucleic acid comprises a first transcriptional terminator sequence (TT1), wherein the U4 comprises a fourth pair of recombinase recognition sequences (RRS4) in the same orientation with respect to each other, wherein each of the RRS4 flanks each side of a second TG2,   further comprising (i) a fifth recombination unit (U5) positioned downstream of the U1 and comprising a fifth pair of recombinase recognition sequences (RRS5) in the same orientation with respect to each other and a fifth nucleic acid sequence, each of the RRS5 flanking each side of the fifth nucleic acid sequence, the fifth nucleic acid sequence comprising a sixth recombination unit (U6) and a third TG2 and a second TG1 connected in series and positioned downstream of the U6, the U6 comprising a sixth pair of recombinase recognition sequences (RRS6) in the same orientation with respect to each other, wherein each of the RRS6 flanks each side of a second transcriptional terminator sequence (TT2); (ii) a seventh recombination unit (U7) positioned downstream of the U5, the U7 comprising a seventh pair of recombinase recognition sequences (RRS7) in the same orientation with respect to each other, wherein each of the RRS7 flanks each side of a fourth TG2; (iii) a third transcriptional terminator sequence (TT3) positioned downstream of the U7,   and wherein the R1 recognizes the RRS1, the R2 recognizes the RRS2, RRS6, and RRS7, and the R3 recognizes the RRS3, RRS4, and RRS5.   
           23. The nucleic acid logic cassette of any one of the above paragraphs, wherein the target gene encodes a reporter molecule, a protein of interest, an RNA of interest, or an enzyme of interest.   24. The nucleic acid logic cassette of paragraph 23, wherein the reporter molecule is selected from the group consisting of β-galactosidase, chloramphenicol acetyltransferase, neomycin phosphotransferase, green fluorescent protein, mCherry, red fluorescent protein, and secreted placental alkaline phosphatase (SEAP), secreted a-amylase (SAMY), firefly luciferase, and  Renilla  luciferase.   25. The nucleic acid logic cassette of any one of the above paragraphs, wherein the R1, R2, and R3 are each a tyrosine recombinase or a serine recombinase.   26. The nucleic acid logic cassette of any one of the above paragraphs, wherein the R1, R2, and       

     R3 are each selected from the group consisting of Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, λ, HK022, HP1, γδ, ParA, Tn3, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, PhiMR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, and HbiF.
         27. The nucleic acid logic cassette of any one of the above paragraphs, wherein each pair of recombinase recognition sequences is selected from the group consisting of loxP, loxN, lox 511, lox 5171, lox 2272, M2, M3, M7, M11, lox71, lox66, FRT, rox, SloxM1, VloxP, vox, B3RT, KDRT, F3, F14, attB/P, F5, F13, Vlox2272, Slox2272, SloxP, RSRT, and B2RT.   28. The nucleic acid logic cassette of any one of the above paragraphs, wherein the promoter is an inducible promoter.   29. The nucleic acid logic cassette of any one of the above paragraphs, wherein the promoter is an constitutive promoter.   30. A nucleic acid logic system comprising:
           (i) at least one nucleic acid sequence encoding at least one inducible promoter operatively linked to at least one recombinase, whereby expression of the at least one recombinase provides an input to a nucleic acid logic cassette adapted to perform a logic function as a function of the input, and   (ii) the nucleic acid logic cassette of any one of paragraphs 1-29.   
           31. The nucleic acid logic system of paragraph 30, wherein the logic function is selected from the group consisting of NOR, OR, AND, NAND, A IMPLY B, B IMPLY A, A NIMPLY B, B NIMPLY A, XOR, XNOR, decoder, half adder, half subtractor, half adder-subtractor, full adder, full subtractor, Feynman gate, logic selector, Ripple carry adder, array multiplier, arithmetic logic unit, and any combinations thereof.   32. A mammalian cell containing the nucleic acid logic cassette of any one of paragraphs 1-29.   33. A mammalian cell containing a nucleic acid logic system of paragraph 30 or 31.   34. A mammalian immune cell containing a nucleic acid logic cassette, the cassette comprising:
           (i) a nucleic acid sequence encoding a mammalian promoter;   (ii) a first recombination unit (U1) comprising a first pair of recombinase recognition sequences (RRS1) for a first recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter,
               whereby when the R1 recognizes the RRS1, the first nucleic acid sequence is excised when the RRS1 are in the same orientation or is inverted when the RRS1 are in the inverse orientation;   
               (iii) a second recombination unit (U2) comprising a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in the same orientation or in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1,
               whereby when the R2 recognizes the RRS2, the second nucleic acid sequence is excised when the RRS2 are in the same orientation or is inverted when the RRS2 are in the inverse orientation,   
               
            wherein the first or second nucleic acid sequence comprises a target gene, and wherein presence or absence of at least one of the R1 and R2 operatively links the promoter to at least one of the first and second nucleic acid sequence, thereby driving expression of the first or second nucleic acid sequence.   35. The mammalian cell of paragraph 34, wherein the immune cell is a T cell.   36. The mammalian cell of paragraph 34, wherein the immune cell is a B cell.   37. The mammalian cell of any one of paragraphs 34-36, wherein the target gene encodes a reporter molecule, a protein of interest, an RNA of interest, or an enzyme of interest.   38. The mammalian cell of paragraph 37, wherein the reporter molecule is selected from the group consisting of β-galactosidase, chloramphenicol acetyltransferase, neomycin phosphotransferase, green fluorescent protein, mCherry, red fluorescent protein, secreted placental alkaline phosphatase (SEAP), secreted a-amylase (SAMY), firefly luciferase, and  Renilla  luciferase.   39. The mammalian cell of any one of paragraphs 34-38, wherein the R1 and R2 are each a tyrosine recombinase or a serine recombinase.   40. The mammalian cell of any one of paragraphs 34-39, wherein the R1 and R2 are each selected from the group consisting of Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, λ, HK022, HP1, γδ, ParA, Tn3, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, PhiMR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, and HbiF.   41. The mammalian cell of any one of paragraphs 34-40, wherein the RRS1 and RRS2 are each selected from the group consisting of loxP, loxN, lox 511, lox 5171, lox 2272, M2, M3, M7, M11, lox71, lox66, FRT, rox, SloxM1, VloxP, vox, B3RT, KDRT, F3, F14, attB/P, F5, F13, Vlox2272, Slox2272, SloxP, RSRT, and B2RT.   42. The mammalian cell of any one of paragraphs 34-41, wherein the nucleic acid logic cassette is adapted to perform a logic function.   43. The mammalian cell of paragraph 42, wherein the logic function is selected from the group consisting of NOR, OR, AND, NAND, A IMPLY B, B IMPLY A, A NIMPLY B, B NIMPLY A, XOR, XNOR, decoder, half adder, half subtractor, half adder-subtractor, full adder, full subtractor, Feynman gate, logic selector, Ripple carry adder, array multiplier, arithmetic logic unit, and any combinations thereof.   44. A switch operable in a mammalian immune cell, comprising:
           (i) a nucleic acid sequence encoding a mammalian promoter;   (ii) a first pair of recombinase recognition sequences (RRS1) for a first recombinase (R1), wherein each of the RRS1 flanks each side of a first nucleic acid sequence, and wherein the RRS1 are in an inverse orientation with respect to each other, and wherein at least one of the RRS1 is positioned downstream of the promoter;   (iii) a second pair of recombinase recognition sequences (RRS2) for a second recombinase (R2), wherein each of the RRS2 flanks each side of a second nucleic acid sequence, and wherein the RRS2 are in an inverse orientation with respect to each other, and wherein at least one of the RRS2 is positioned downstream of at least one of the RRS1; and   (iv) at least one target gene positioned downstream of at least one of the RRS1, whereby expression of the target gene is controlled by the presence or absence of at least one of the R1 and R2.   
           45. The switch of paragraph 44, wherein the immune cell is a B cell.   46. The switch of paragraph 44, wherein the immune cell is a T cell.   47. The switch of any one of paragraphs 44-46, wherein the target gene encodes a chimeric antigen receptor (CAR).   48. The switch of any one of paragraphs 44-47, wherein the switch is selected from the group consisting of an On switch, an Off switch, an Expression Level switch, an Affinity Switch, and a Target switch.   49. The switch of paragraph 48, wherein the switch is the Off switch, wherein the first nucleic acid sequence comprises one of the RRS2 and the target gene, and wherein the second nucleic acid sequence comprises the target gene and one of the RRS1, and wherein the target gene is in the same orientation as the promoter, whereby in the presence of the R2 and then the R1, the target gene is inverted, thereby turning off the expression of the target gene.   50. The switch of paragraph 48, wherein the switch is the On switch, wherein the first nucleic acid sequence comprises one of the RRS2 and the target gene, and wherein the second nucleic acid sequence comprises the target gene and one of the RRS1, and wherein the target gene is in an inverted orientation, whereby in the presence of the R2 and then the R1, the target gene is inverted, thereby turning on the expression of the target gene.   51. The switch of paragraph 48, wherein the switch is the Expression Level switch, wherein the promoter is in an inverted orientation, wherein the first nucleic acid sequence comprises the promoter and one of the RRS2, and wherein the second nucleic acid sequence comprises the promoter and one of the RRS1, and wherein the target gene is positioned downstream of the RRS1 and RRS2, whereby in the presence of the R1 and R2, the promoter is inverted, thereby turning on the expression of the target gene.   52. The switch of paragraph 48, wherein the switch is the Target switch, wherein the first nucleic acid sequence comprises a first target gene in the same orientation as the promoter, one of the RRS2, and a second target gene in an inverted orientation, and wherein the second nucleic acid sequence comprises the second target gene and one of the RRS1, whereby in the presence of the R1 and R2, the second target gene is operatively linked to the promoter, thereby turning on the expression of the second target gene and turning off the expression of the first target gene.   53. The switch of paragraph 52, wherein the switch is the Affinity switch, wherein the first target gene encodes a low-affinity CAR, and wherein the second target gene encodes a high-affinity CAR.   54. The switch of any one of paragraphs 44-53, wherein the R1 and R2 are each a tyrosine recombinase or a serine recombinase.   55. The switch of any one of paragraphs 44-54, wherein the R1 and R2 are each selected from the group consisting of Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, λ, HK022, HP1, γδ, ParA, Tn3, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, PhiMR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, and HbiF.   56. The switch of any one of paragraphs 44-55, wherein the RRS1 and RRS2 are each selected from the group consisting of loxP, loxN, lox 511, lox 5171, lox 2272, M2, M3, M7, M11, lox71, lox66, FRT, rox, SloxM1, VloxP, vox, B3RT, KDRT, F3, F14, attB/P, F5, F13, Vlox2272, Slox2272, SloxP, RSRT, and B2RT.   57. The switch of any one of paragraphs 44-56, adapted to be activated by a compound selected from the group consisting of doxycycline, tamoxifen, rapamycin, and abscisic acid.   58. A method of treating cancer in a subject, the method comprising
           (i) incorporating a switch of any one of paragraphs 44-57 into T cells, thereby permitting the T cells to express the switch; and   (ii) administering the T cells to the subject.   
           59. The method of paragraph 58, further comprising expanding the T cells prior to the administering.   60. The method of paragraph 58 or 59, further comprising administering a compound selected from the group consisting of doxycycline, tamoxifen, rapamycin, and abscisic acid to activate the switch.       

     Definitions 
     Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. 
     As used herein, the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. 
     As used herein, the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. 
     As used herein, the term “cassette” refers to a nucleic acid molecule, or a fragment thereof, that can be introduced to a host cell and incorporated into the host cell&#39;s genome. A cassette can comprise a target gene. A cassette can be an isolated nucleotide fragment, e.g. a dsDNA or can be comprised by a vector, e.g. a plasmid, cosmid, and/or viral vector. In some embodiments, a cassette is a single nucleic acid construct, e.g., an engineered nucleic acid sequence. 
     As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA. 
     As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein. 
     Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of lupus nephritis. A subject can be male or female. 
     As used herein, the term “administering,” refers to the placement of a compound or a cell into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Any appropriate route which results in an effective treatment in the subject can be used. 
     Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intrahepatic, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. The administration can be systemic or local. 
     A cell for use with the cassettes and switches described herein can be any cell or host cell. As defined herein, a “cell” or “cellular system” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular. A “natural cell,” as defined herein, refers to any prokaryotic or eukaryotic cell found naturally. A “prokaryotic cell” can comprise a cell envelope and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. 
     In some embodiments, the cell is a eukaryotic cell, preferably a mammalian cell. A eukaryotic cell comprises membrane-bound compartments in which specific metabolic activities take place, such as a nucleus. In other embodiments, the cell or cellular system is an artificial or synthetic cell. As defined herein, an “artificial cell” or a “synthetic cell” is a minimal cell formed from artificial parts that can do many things a natural cell can do, such as transcribe and translate proteins and generate ATP. 
     Cells of use in the various aspects described herein upon transformation or transfection with the cassettes or switches described herein include any cell that is capable of supporting the activation and expression of the molecular circuits. In some embodiments of the aspects described herein, a cell can be from any organism or multi-cell organism. Examples of eukaryotic cells that can be useful in aspects described herein include eukaryotic cells selected from, e.g., mammalian, insect, yeast, or plant cells. The molecular circuits described herein can be introduced into a variety of cells including, e.g., fungal, plant, or animal (nematode, insect, plant, bird, reptile, or mammal (e.g., a mouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale, monkey, or human)). The cells can be primary cells, immortalized cells, stem cells, or transformed cells. In some preferred embodiments, the cells comprise stem cells. Expression vectors for the components of the molecular circuit will generally have a promoter and/or an enhancer suitable for expression in a particular host cell of interest. The present invention contemplates the use of any such vertebrate cells for the molecular circuits, including, but not limited to, reproductive cells including sperm, ova and embryonic cells, and non-reproductive cells, such as kidney, lung, spleen, lymphoid, cardiac, gastric, intestinal, pancreatic, muscle, bone, neural, brain, and epithelial cells. 
     As used herein, the term “gene” refers to a nucleic acid sequence comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. A “gene” refers to coding sequence of a gene product, as well as non-coding regions of the gene product, including 5′UTR and 3′UTR regions, introns and the promoter of the gene product. These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid sequence can encompass a double-stranded molecule or a double-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a molecule. As used herein, a single stranded nucleic acid can be denoted by the prefix “ss”, a double stranded nucleic acid by the prefix “ds”, and a triple stranded nucleic acid by the prefix “ts.” 
     The term “expression” as used herein refers to the biosynthesis of a gene product, preferably to the transcription and/or translation of a nucleotide sequence, for example an endogenous gene or a heterologous gene, in a cell. For example, in the case of a heterologous nucleic acid sequence, expression involves transcription of the heterologous nucleic acid sequence into mRNA and, optionally, the subsequent translation of mRNA into one or more polypeptides. Expression also refers to biosynthesis of a microRNA or RNAi molecule, which refers to expression and transcription of an RNAi agent such as siRNA, shRNA, and antisense DNA but does not require translation to polypeptide sequences. The term “expression construct” and “nucleic acid construct” as used herein are synonyms and refer to a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence, such as the heterologous target gene sequence in an appropriate host cell (e.g., a prokaryotic cell, eukaryotic cell, or mammalian cell). If translation of the desired heterologous target gene is required, it also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region can code for a protein of interest but can also code for a functional RNA of interest, for example, microRNA, microRNA target sequence, antisense RNA, dsRNA, or a nontranslated RNA, in the sense or antisense direction. The nucleic acid construct as disclosed herein can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp &amp; Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway&#39;s Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor &amp; Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin&#39;s Genes XI, published by Jones &amp; Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. 
     Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages may mean±1% of the value being referred to. For example, about 100 means from 99 to 101. 
     Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” 
     Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein. 
     All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology disclosed herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. 
     The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are disclosed herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments disclosed herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. 
     Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. 
     EXAMPLES 
     The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The technology disclosed herein is further illustrated by the following examples which in no way should be construed as being further limiting. 
     Materials and Methods: 
     DNA Assembly. 
     All constructs were transformed and maintained in Top10 competent cells (Life technologies) at 37° C. or 30° C. prior to miniprep (Epoch Life Sciences) or midiprep (Macherey Nagel). All plasmids were created using standard molecular biology practices of ligation, digestion and transformation, in addition to Gibson isothermal assembly and Unique Nucleotide Sequence Guided assembly (a modular extension of Gibson assembly), where DNA fragments that are to be connected to each other are flanked by short homology sequences and are then fused together by a one-pot isothermal digestion, polymerization, and ligation reaction. 
     Construction Details of DNA Assembly. 
     Most genetic reporters were created using an extension of Gibson isothermal assembly called Unique Nucleotide Sequence (UNS) Guided Assembly. This strategy permits a modular, easy, efficient and fast framework for construction of DNA. In UNS-guided assembly, the homology sequences have been standardized (e.g. U1, U2, U3 . . . UK) and have been computationally optimized for proper assembly (reduction of hairpins, sequence homology, and GC tracts) and ease of use (no start codons or useful restriction sites). Genetic cassettes that are to be connected are cloned into part vectors, which contain the UNSes that surround the insert. Restriction digests and gel purifications are then performed to isolate the cassettes flanked with the UNSes. Finally, these products are joined to a linearized destination vector via Gibson isothermal assembly (see also  FIGS. 9 and 10 ). 
     Transient DNA Transfection into HEK293FT Cells. 
     DNA was transfected into the human embryonic kidney cell line (HEK293FT) using a polyethylenimine (PEI) protocol. Cells were plated onto 48- or 96-well plates the day prior to transfection, such that the cells were 50-70% confluent the day of transfection. Cells were kept in a humidified incubator at 37° C. and 5% CO 2  and maintained in DMEM medium (Corning) with 10% Heat Inactivated Fetal Bovine Serum (Life technologies), 50 U.I./mL penicillin/50 μg/mL streptomycin (Corning), 2 mM glutamine (Corning) and 1 mM sodium pyruvate (Lonza). 
     Flow Cytometry. 
     Two days post-transfection and after trypsinization (0.05% T sin/0.53 mM EDTA, Corning) and resuspension, all cell populations were analyzed using a Becton Dickinson (BD) LSRFortessa SORP flow cytometer with HTS, except for data in  FIG. 2B  and  FIG. 4 , which were recorded on a BD LSRII. The LSRFortessa was equipped for detection of EGFP (488 nm laser, 530/30 emission filter, 505 longpass filter), tagBFP (405 nm laser, 450/50 emission filter), mCherry or mRuby2 (561 nm, 610/20 emission filter, 595 longpass filter), iRFP-720 (637 nm laser, 730/45 emission filter, 685 longpass filter) and LSS-mOrange (405 nm, 610/20 emission filter, 535 longpass filter). The LSRII was similarly equipped for detection of EGFP, tagBFP, mCherry and mRuby2, but with additional channels for iRFP-720 (633 nm laser, 720/40 emission filter, 710 longpass filter) and LSS-mOrange (405 nm, 590/35 emission filter, 505 longpass filter). 
     Transient Transfection into HEK293 Cells Using Polyethylenimine. 
     Polyethylenimine (PEI) stocks were made from linear polyethylenimine (Polysciences 23966-2) and were dissolved at a concentration of 0.323 g/L in deionized water with the assistance of concentrated hydrochloric acid and sodium hydroxide and then filtered sterilized (0.24 m). PEI stocks were stored at −80° C. until use and warmed to room temperature prior to usage. For 48-well plate transfections, DNA (1000 ng) was dissolved and brought up to a volume of 50 μL using 0.15 M sodium chloride (NaCl, Fisher Scientific). DNA-NaCl solutions were then mixed with 50 μL of a PEI-NaCl mixture (8 uL PEI: 42 uL NaCl). These solutions were then incubated at room temperature for ten minutes and 25 μL was carefully dropped into individual wells of HEK293FT cells (250 ng DNA/well). Similarly for 96-well plate transfections, DNA (500 ng) was dissolved and brought up to a volume of 25 μL using 0.15 M sodium chloride (NaCl). DNA-NaCl solutions were then mixed with 25 μL of a PEI-NaCl (4 uL PEI: 21 uL NaCl) mixture. These solutions were then incubated at room temperature for ten minutes and 10 μL was carefully dropped into individual wells of HEK293FT cells (100 ng DNA/well). 
     Example 1 
     A useful feature of site-specific recombinases is that they can act as transcription activators or repressors depending on the placement of recombination sites. For instance, buffer (BUF) gates permit recombinase-mediated gene expression through tyrosine recombinase-mediated excision of a transcription terminator or serine integrase-mediated inversion ( FIG. 1A ). Conversely, NOT gates function to terminate gene expression via placement of recombination sites around a gene of interest and elimination of expression through tyrosine recombinase-mediated excision or serine integrase inversion. Increasingly, optogenetics, animal disease models, and study of stem cell generation and differentiation have relied on recombinase-based strategies to achieve tissue-specific gene expression or knockout, utilizing a tissue-specific promoter (TSP) to drive the recombinase expression in combination with a BUF or NOT gate containing a gene of interest (GOI) 18,20-23 . Although these tools have been instrumental in many studies, targeting a specific tissue with only one TSP often resulted in off-target expression 2,24 . To improve specificity, 2-input AND logical integration of two recombinases driven from two separate TSP&#39;s has been employed. Realizing that many cell types cannot be adequately specified with a 2-input AND gate, the inventors developed a powerful yet intuitive platform for rapidly creating genetic multi-input logic computation circuits in mammalian cells. 
     Multi-input recombinase-based logic gates require robust and orthogonal genetic components. Here, twelve recombinases, including both tyrosine recombinase and serine integrase families were tested for activity and orthogonality in a human embryonic kidney cell line (HEK293FT). Through transient transfection of recombinases and their BUF gates, ten of the enzymes were found to be highly active and sufficiently orthogonal to each other for the desired circuit design ( FIG. 1B ). 
     Surprisingly, some of the reportedly orthogonal recombinases, such as Dre, Cre, Vika, and VCre 25-28  showed cross reactivity in a dose-dependent manner ( FIG. 6 ). Therefore, when using these recombinases for circuit design, a lower dose was required to minimize crosstalk. By applying these properties to Cre and Flp, two of the most commonly used recombinases in mammalian genetics literature, the inventors created all sixteen possible two-input Boolean logic gates ( FIG. 7 ). Note that the 2-input AND is created by placing two transcription termination sequences in tandem. Due to the orthogonality of recombinases, it becomes possible to generate multi-input AND gates simply by placing more termination sequences in tandem between a promoter and GFP. Indeed, the inventors developed a 6-input AND gate that expresses GFP upon the excision of four termination sequences by tyrosine recombinases and the inversion of the EF1α promoter and GFP by two serine integrases ( FIG. 1C ). In contrast to earlier multi-layer 4-input AND gate work in  E. coli   13 , this multi-input AND gate was on a single layer, which could facilitate implementation in mammalian genetic systems. 
     A key element that allows complex computation on a single transcriptional unit is the usage of heterospecific recombination sites. Heterospecific sites, such as loxP and lox2272, differ from one another by only a few base pairs, 29  but they retain DNA excision capability in the presence of Cre, as long as two of the same sites are present, i.e. loxP with loxP. This feature allows the excision of more than one non-connected region of DNA simultaneously. 
     Example 2 
     The inventors exploited these characteristics to establish a platform for generation of N-input order-independent logic gates called Boolean Logic and Arithmetic through DNA Excision (BLADE). All logic computation for BLADE circuits is done on a single transcriptional layer and circuit construction is based on a BLADE template. N-input BLADE templates feature 2 N  distinct regions of DNA termed addresses (Z). Each address is accessible only via a specific combination of inputs. For example, the 2-input BLADE template contains four addresses, Z=Z AB =Z 00 , Z 10 , Z 01 , and Z 11 , corresponding to each state of A and B inputs ( FIG. 2A ). Each address contains one output function, which ranges from having zero outputs (transcription terminator), through arbitrary numbers of outputs separated by ribosomal skip sequences (2A), to Boolean functions like BUF. 
     For initial characterization of the 2-input BLADE platform, single-output functions were tested. The inventors constructed a 2-input decoder circuit in which each address coincides with a distinct output function. Blue, green, infrared and red fluorescent proteins (tagBFP, EGFP, iRFP720, and mRuby2, respectively) were placed into the four addresses of the 2-input BLADE template ( FIG. 2B ). The circuit performed as predicted with strong fluorescent protein expression for each state of inputs, though there was a small degree of spill over between states, due to transient, basal gene expression that occurred prior to and following the recombination reactions. This circuit may be particularly useful as it could permit targeted expression of four different genes in four distinct cell types simultaneously using only two tissue-specific promoters. Furthermore, to test the robustness of this circuit design on a large scale, the inventors then produced a library of &gt;100 circuits with up to two inputs and outputs using Unique Nucleotide Sequence-Guided Assembly, a modular and rapid Gibson isothermal assembly method 30,31  ( FIG. 3A  and  FIG. 9 ). Of note, are three 2-input devices: the half adder (Gate 11) and half subtractor (Gate 17), which both perform 2-input arithmetic and the Controlled Not Feynman gate (Gate 55), which exhibits interesting logic behavior. 
     One important class of devices found in electronics is Field-Programmable Read-Only Memory (FPROM). The input-output behavior of these circuits can be configured in the field post-manufacturing, allowing users to customize their devices at a later time. The inventors built a FPROM device termed a Boolean Logic Look-up Table (LUT) that is based on placing BUF gates into the four addresses of the 2-input BLADE template ( FIG. 4 ). This circuit has two data inputs, A and B, and four select inputs, S 1 , S 2 , S 3 , and S 4 . Each select input can control which buffer gates are GFP ON or OFF. Thus, each combination of select inputs configures the device to a specific Boolean logic gate with up to two inputs and one output. For instance, an OR function can be achieved using select inputs S 2 , S 3 , and S 4 , keeping address Z 00  GFP OFF and setting addresses Z 10 , Z 01 , and Z 11  GFP ON. This circuit behaves as expected in mammalian cells. To illustrate the flexibility of the gene circuit platform in terms of recombinase choices, the inventors created an alternative Boolean Logic LUT ( FIGS. 8A-8B ). 
     Extending the BLADE framework further, the inventors then developed a 3-input BLADE template for constructing sophisticated arithmetic functions in human cells ( FIG. 5A ). This template responds to three inputs (Cre, Flp, and VCre) and contains eight addresses for expression of up to eight distinct output functions. This design utilizes three different heterospecific sites for Cre and Flp, but just one site for VCre. Three 3-input-2-ouput arithmetic computational circuits were made and tested in mammalian cells from the 3-input BLADE template ( FIG. 5B ). The full adder and full subtractor can perform either binary addition or subtraction of three 1-bit inputs, respectively. Furthermore, the half adder-subtractor is an arithmetic FPROM circuit that can compute addition or subtraction on two data inputs, A and B, depending on the presence of one select input C. 
     Example 3 
     The development of multi-input logic gates requires robust and orthogonal recombinases. The inventors tested more than 10 different recombinases, including both the tyrosine and serine recombinase family in HEK 293T, a human embryonic kidney cell line. To test the tyrosine recombinases, a reporter plasmid for every recombinase was constructed where the corresponding recombination sites flanking a neomycin resistance gene followed by a SV40 polyA sequence (ref) is placed between a CAG promoter and a GFP. The neomycin and SV40 polyA sequence forms a transcriptional stop cassette and prevent the transcription of GFP. When the proper recombinase is expressed, it will bind to the two recombination sites and excise the stop cassette between the recombination sites ( FIG. 11A ). This reporter construct is also known as a buffer gate and will be used in other circuit design detailed later in this text. For serine recombinases, the recombination sites are placed between a GFP that is in antisense direction. The expression of the corresponding serine recombinase inverts the DNA fragment between the recombination sites, thus inverting the GFP into the sense direction and allowing GFP to be expressed ( FIG. 11A ). Through transient transfection of the recombinase and the reporter plasmid, most of the enzymes tested are found to be highly active and sufficiently orthogonal for the desired circuit design ( FIG. 11B ). 
     A unique feature of recombinase is that it can act as both a transcription initiator (i.e., activators) and suppressors (i.e. repressors), depending on the placement of the recombination site. For instance, by placing a stop cassette between recombination sites, the expression of the corresponding recombinase excises the transcription stop sequences and allows transcription to occur. Similarly, flanking the gene of interest with recombinase allow the recombinase to remove the gene, thus serve as suppressor of gene expression. Using these unique properties of recombinase, the inventors successfully created all 16 possible 2-Input Boolean logic gates ( FIG. 7 ) using the most commonly used recombinases in mammalian genetics, Cre and Flp. Note that the AND is created by placing two stop cassette in tandem. Because of the othogonality of recombinases, it becomes possible to generate multi-Input AND gates simply by placing more stop cassettes in tandem between a GFP and the promoter. Indeed, the inventors have created several version of 4- and 6-Input AND gates using different enzymes and one 8-Input AND gates ( FIGS. 12A-12C ). In contrast to earlier multi-layer 4-Input AND gate work in  E. coli , these multi-Input AND gates are single layer and will greatly facilitate implementation in mammalian genetic systems. 
     Any 2-Input logic gates has 4 distinct states. When each state generates either 0 or 1, they form the traditional 2-Input Boolean logic gates mentioned above. However, when each state generates a distinct output, this creates a 2-4 decorder ( FIG. 13A ). A genetically encodable 2-4 decoder will allow targeted gene expression in 4 distinct cell types simultaneously using just two recombinases input. In addition, realizing the advantage of the single transcription unit design for implementation in mammalian system, the inventors therefore sought to create a recombinase-based decorder in a single transcription unit. To achieve this goal, the inventors designed a decoder circuit where there are 4 distinct DNA fragment slots downstream of the promoter. Each slot is surrounded by different permutation of recombination sites configurations and corresponds to a unique state. A key enabling element that allows this design to work is the heterospecific recombination sites available for some of the recombinase, such as Cre and Flp. Heterospecific sites, such as loxP and lox2272, differ from each other by a few base pairs. However, they retain the ability to excise DNA in the presence of Cre, but will only react with the same type of site—loxP with loxP and lox2272 with lox2272. This feature allows the excision of more than one non-connected slot simultaneously. Without using heterospecific sites, decoder circuit can still be implemented, albeit requires several transcription unit. 
     To illustrate how this design work, consider the first slot (Z 0,0 ), which is the closest to the promoter. The Z00 correspond to a state where no recombinase is expressed (0,0). If the slot Z00 contains a protein coding sequence, then that gene will be expressed. Gene expression from the other slots downstream of Z00 will be blocked by the presence of Z00. In the presence of recombinase A, which correspond to state (1,0), slot Z0,0 and Z0,1 will be removed, thus moving slot Z1,0 directly downstream of the promoter and allow gene expression of slot Z1,0 and only Z1,0, to occur. Similarly, when recombinase B is presence, which correspond to state (0,1), slot Z00 and Z10 are removed, allowing Z01 to be moved directly downstream of the promoter. Finally, when both recombinases are expressed, slot Z00, Z01, Z10 are all excised, thus placing Z11 downstream of the promoter unobstructed by other slots. Note that each slot is not limited to the expression of just one gene/output. For example, an IRES or 2A type of sequence can be used to link multiple genes together in one slot, thus allowing arbitrary number of outputs. To demonstrate this decoder design experimentally, the inventors placed a stop cassette, mRuby2, iRFP, and GFP into slot Z00, Z10, Z01, and Z11 respectively, and transfected into HEK293T along with Cre and Flp expressing plasmids. The decorder output is highly digital with a signal to noise ratio over 100 for each state ( FIG. 13A ). 
     This 2-4 decoder design theoretically allow the generation of all possible 2-Input n-output devices in a single layer. Some of the more important 2-Input devices are the half adder and half subtractor, which are 2-Input 2-Output devices that can perform arithmetic. A half adder generates an output in the Sum (S) when either one of the input is presence. When both inputs are presence, it generates a carry out signal (Cout) and no S. To generate a half-adder, the inventors placed a stop cassette in the Z00 slot, a GFP in Z10 and Z01, and a GFP-2A-mCherry cassette in Z11 ( FIG. 13B ). In this case, the GFP is the S and mCherry is the Cout. Similar to the decoder, half adder performs as expected in mammalian cell with high signal to noise ratio ( FIG. 4 b   ). To create the half subtractor, a stop cassette is placed in the Z00 slot, a GFP in Z01, and a GFP-P2A-mCherry in Z10. Since there is no output in both the S and Cout, therefore nothing is required in the Z11 slot. Again, the half subtractor behaved as expected ( FIG. 13C ). 
     If heterospecific sites are not available for the enzyme of choice, it is still possible to create half adder or half subtractor using multiple transcription units. The inventors noticed that half adder or half subtractor can be composed from some of the simpler 2-Input 1 output Boolean logic gates. For example, a half adder is composed of an A-NIMPLYB and BNIMPLY with a GFP as output and a AND gate with mCherry as output. The inventors can achieve a half adder simply by co-transfecting three plasmids, each containing a transcription unit for a logic gate. The inventors can also combine the two NIMPLY logic gates into one unit to form a XOR gate and reduces the design into a two transcription unit system. Note that although it may be easier to implement a multi-transcription unit design in transient transfection because one can mix different number of plasmids readily, single transcription unit will facilitate integration into genome. 
     To test whether one can construct more sophisticated circuits, the inventors developed a Full-Adder, a 3-Input 2-output logic gates, using Cre, Flp and Vcre as input. Similar to the half-adder, a full adder is one of the permutations of the 3-8 decoder. A 3-8 decoder has 8 unique states, thus requires 8 different slots. This design requires 3 different heterospecific sites for Cre and Flp, but just one site for Vcre. Again, each slot corresponds to a specific state. To achieve a full adder, the inventors filled the slots with either a stop cassette, GFP, mCherry or GFP-2A-mCherry, depending on the out of the full adder truth table. This single layer design displayed the expected logic output in mammalian cells ( FIG. 13D ). 
     An important class of circuit found in modern computer is a Programmable Read Only Memory (PROM) device where depending on the input selection signal, the circuit will adopt one of the 16 2-Input 1 Output Boolean logic gates. In essence, this is a 6-Input 1-Output logic gate. To create the PROM, the Cre-Flp decoder framework is employed. Each slot is created with recombination sites from Cre and Flp. However, in each slot, a buffer gate for different selector input recombinase is inserted. In the present version, buffer gate for Dre, Vox, VCre and B3 is in the slot Z00, Z10, Z01, and Z11 respectively. Therefore, the selector input will dictate whether a stop cassette or a GFP is dominant in that slot. For instance, within a decoder framework, an OR can be achieved with a stop cassette in the Z00 slot and a GFP in the other three slots. Therefore, in the presence of Vox, VCre, and B3, slot Z10, Z01, and Z11 will have GFP while Z00 will still have a stop cassette, thus generating the OR gate. As shown in  FIG. 8A , the logic gate selector can display the output logic of all 16 Boolean logic gates depending on the selector input recombinase. 
     In electronic circuit design, many of the more sophisticated logic circuits were created by connecting several simpler circuits together, often times in multiple layers. This is a robust design strategy in electronic, because the connection between different device is easy to implement and the resulting performance is predictable. However, following this strategy from electronic in synthetic biology has proven to be difficult because of the lack of understanding of how different components will behave when connected together. Large efforts are underway to provide detail characterization of components and develop standard to allow better predictability of the interoperability of different parts. 
     Here, a complementary approach is provided where a single layer platform can be used to create a large variety of complex circuits. This platform is highly robust, such that all of the inventors demonstrated that designed and developed circuits performed as expected on first attempt without iteration. To characterize the robustness of this platform, a library of 2-Input 2-Output circuit can be used by using a combinatorial Gibson assembly strategy ( FIG. 9 ). Using the 2-4 decoder framework as the basis, 4 parts can be constructed for each slot—stop cassette, GFP, mCherry, and GFP-2A-mCherry. Since the 2-4 decoder has 4 slots, a total of 16 components can be used. Then a combinatorial assembly strategy can be employed to create all 256 possible 2-Input 2-Output circuits. Characterizing the percentage of the 256 circuits that behave as expected will characterize the robustness of the platform. Similar libraries can be created for all other n-Input m-Output circuit, thus providing a foundation to generate an enormous amount of functionally distinct circuits. 
     All the circuits designed in the work are single use system. While this single use system as disclosed herein is sufficient for many biotechnological applications, such as in animal model development, it can also be constructed to use a reversible system for other occasions. While both tyrosine and serine recombinase can perform excision and inversion, only serine recombinase can perform stable inversion. More importantly, some serine recombinase has a complementary directionality factor that when expressed together with the recombinase, will revert the inversion generated by the recombinase back to the original configuration. Using this property, the inventors also designed a decoder frame work based on inversion of serine recombinase, and depending on the presence of directionality factor or not, can revert the system back to the original state ( FIGS. 14A-14B ). Similar to the tyrosine recombinase design, such decoder framework also relies on the availability of heterospecific sites. 
     Example 4 
     In electronic circuit design, connecting several simple circuits together, often times in multiple layers, was applied in order to generate many sophisticated computational circuits. This is a robust design strategy in electronics, because the connections between different components are easy to implement and the resulting performance is predictable. However, applying this strategy from electrical engineering to synthetic biology has proven to be difficult because of the lack of a complete understanding of how different components will behave when connected together. Efforts are underway to provide detailed characterization of components and develop standards to allow for better predictability of the interoperability of different parts 32-34 . Furthermore, multi-layer design requires more orthogonal biological “wires”, which drives effort toward parts development 35-38  at the expense of circuit design. Here, the inventors have demonstrated a simple and flexible approach in which a single layer platform can be used to create a large variety of complex circuits. This platform is highly robust, such that the developed circuits performed as expected on the first attempt without iteration. Coupled with the recent discoveries of many orthogonal serine integrases 39 , the BLADE platform provides the foundation for creating advanced genetic computational devices in living cells and opens the doors for complex animal model development. 
     Example 5 
     
       
         
           
               
             
               
                 TABLE 42-1 
               
             
            
               
                   
               
               
                 Setup of buffer gates and recombinase expression plasmids for recombinase 
               
               
                 cross-reactivity table as detailed in FIG. 1B, in addition to co-transfection 
               
               
                 with 62.5 ng pCAG-tagBFP (pBW462) and 62.5 ng pCAG-FALSE (pBW363). 
               
            
           
           
               
               
            
               
                   
                 Reporter 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 loxP 
                 FRT 
                 rox 
                 SloxM1 
                 VloxP 
                 vox 
                 B3RT 
                 KDRT 
               
               
                 Enz 
                 Plasmid ID 
                 pBW338 
                 pBW339 
                 pBW364 
                 pBW271 
                 pBW273 
                 pBW275 
                 pBW276 
                 pBW277 
               
               
                   
               
               
                 iCre 
                 pBW390 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 FlpO 
                 pBW391 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 DreO 
                 pBW431 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 SCre 
                 pBW432 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 VCre 
                 pBW433 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 Vika 
                 pBW434 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 B3 
                 pBW435 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 KD 
                 pBW436 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 B2 
                 pBW437 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 R 
                 pBW438 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 ϕC31 
                 pBW440 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 bxb1 
                 pBW439 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 None 
                 pBW363 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE 42-2 
               
             
            
               
                   
               
               
                   
                 Reporter 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 ϕC31 
                 bxb1 
                   
               
               
                   
                   
                 B2RT 
                 RSRT 
                 attB/P 
                 attB/P 
                 None 
               
               
                 Enz 
                 Plasmid ID 
                 pBW278 
                 pBW279 
                 pBW409 
                 pBW406 
                 pBW363 
               
               
                   
               
               
                 iCre 
                 pBW390 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 FlpO 
                 pBW391 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 DreO 
                 pBW431 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 SCre 
                 pBW432 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 VCre 
                 pBW433 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 Vika 
                 pBW434 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 B3 
                 pBW435 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 KD 
                 pBW436 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 B2 
                 pBW437 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 R 
                 pBW438 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 ϕC31 
                 pBW440 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 bxb1 
                 pBW439 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                 None 
                 pBW363 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
                 62.5 ng 
               
               
                   
                   
                 ea. 
                 ea. 
                 ea. 
                 ea. 
                 ea. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 Transient transfection setup of six-input AND gate as detailed in FIG. 1D. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 AND GATE, ng 
                 Enzyme 1, ng 
                 Enzyme 2, ng 
                 Enzyme 3, ng 
                 Enzyme 4, ng 
                 Enzyme 5, ng 
                 Enzyme 6, ng 
                 Blank, ng 
                 Marker, ng 
               
               
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
               
               
                 6AND 
                 PhiC31 
                 bxb1 
                 iCre 
                 FlpO 
                 B3 
                 KD 
                 FALSE 
                 tagBFP 
               
               
                 (pBW479) 
                 (pBW440) 
                 (pBW439) 
                 (pBW390) 
                 (pBW391) 
                 (pBW435) 
                 (pBW436) 
                 (pBW363) 
                 (pBW462) 
               
               
                   
               
               
                 150 
                 0 or 12.5 
                 0 or 12.5 
                 0 or 12.5 
                 0 or 12.5 
                 0 or 12.5 
                 0 or 12.5 
                 Fill to 
                 25 
               
               
                   
                   
                   
                   
                   
                   
                   
                 total DNA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 amount = 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 250 ng 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 44 
               
             
            
               
                   
               
               
                 Setup of buffer gates and recombinase expression plasmids for  
               
               
                 recombinase heterospecificity table as detailed in FIG. 1C, in  
               
               
                 addition to co-transfection with 62.5 ng pCAG-tagBFP (pBW462),  
               
               
                 62.5 ng pCAG-FALSE (pBW363), and 62.5 ng corresponding  
               
               
                 recombinase expression plasmid. 
               
               
                 Buffer Gate 
               
            
           
           
               
               
               
            
               
                 Buffer Gate 
                 pBW 
                 ng 
               
               
                   
               
               
                 pCAG-loxP-STOP-loxP-GFP 
                 338 
                 25 
               
               
                 pCAG-lox2272-STOP-lox2272-GFP 
                 427 
                 25 
               
               
                 pCAG-loxN-STOP-loxN-GFP 
                 428 
                 25 
               
               
                 pCAG-loxP-STOP-lox2272-GFP 
                 451 
                 25 
               
               
                 PCAG-loxP-STOP-loxN-GFP 
                 452 
                 25 
               
               
                 pCAG-lox2272-STOP-loxN-GFP 
                 453 
                 25 
               
               
                 pCAG-FRT-STOP-FRT-GFP 
                 339 
                 25 
               
               
                 pCAG-F3-STOP-F3-GFP 
                 429 
                 25 
               
               
                 PCAG-F14-STOP-F14-GFP 
                 430 
                 25 
               
               
                 pCAG-FRT-STOP-F3-GFP 
                 454 
                 25 
               
               
                 pCAG-FRT-STOP-F14-GFP 
                 455 
                 25 
               
               
                 pCAG-F3-STOP-F14-GFP 
                 456 
                 25 
               
               
                 pCAG-VloxP-STOP-VloxP-GFP 
                 273 
                 25 
               
               
                 pCAG-Vlox2272-STOP-Vlox2272-GFP 
                 274 
                 25 
               
               
                 pCAG-VloxP-STOP-Vlox2272-GFP 
                 331 
                 25 
               
               
                   
               
            
           
         
       
     
     Table 45A-45C: Transient transfection setup for 2-input, 1-output logic gates detailed in  FIG. 6 . 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 45A 
               
               
                   
               
               
                   
                   
                 Enzyme 1, ng 
                 Enzyme 2, ng 
                 Blank, ng 
                 Marker, ng 
               
               
                 Re- 
                 Input  
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
               
               
                 porter, 
                 state 
                 iCre 
                 FlpO 
                 FALSE 
                 tagBFP 
               
               
                 ng 
                 A B 
                 (pBW390) 
                 (pBW391) 
                 (pBW363) 
                 (pBW462) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Values 
                 0 0 
                 0 
                 0 
                 50 
                 25 
               
               
                 shown 
                 1 0 
                 25 
                 0 
                 25 
                 25 
               
               
                 below 
                 0 1 
                 0 
                 25 
                 25 
                 25 
               
               
                   
                 1 1 
                 25 
                 25 
                 0 
                 25 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 45B 
               
             
            
               
                   
               
               
                 Logic gate 
               
            
           
           
               
               
               
               
            
               
                   
                 Gate 
                 Plasmid ID 
                 ng 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 HALF ADDER 3 p 
                 pBW344 
                 8.3 
               
               
                   
                   
                 pBW345 
                 8.3 
               
               
                   
                   
                 pBW403 
                 8.3 
               
               
                   
                 HALF ADDER 2 p 
                 pBW448 
                 12.5 
               
               
                   
                   
                 pBW403 
                 12.5 
               
               
                   
                 HALF ADDER 1 p 
                 pBW457 
                 25 
               
               
                   
                 HALF SUBTRACTOR 3 p 
                 pBW344 
                 8.3 
               
               
                   
                   
                 pBW345 
                 8.3 
               
               
                   
                   
                 pBW206 
                 8.3 
               
               
                   
                 HALF SUBTRACTOR 2 p 
                 pBW448 
                 12.5 
               
               
                   
                   
                 pBW206 
                 12.5 
               
               
                   
                 HALF SUBTRACTOR 1 p 
                 pBW570 
                 25 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 45C 
               
             
            
               
                   
               
               
                 Logic gate 
               
            
           
           
               
               
               
               
            
               
                   
                 Gate 
                 Plasmid ID 
                 ng 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 NOR 
                 pBW334 
                 25 
               
               
                   
                 OR 
                 pBW335 
                 25 
               
               
                   
                 AND 
                 pBW336 
                 25 
               
               
                   
                 NAND 
                 pBW337 
                 25 
               
               
                   
                 A 
                 pBW338 
                 25 
               
               
                   
                 B 
                 pBW339 
                 25 
               
               
                   
                 NOTA 
                 pBW340 
                 25 
               
               
                   
                 NOTB 
                 pBW341 
                 25 
               
               
                   
                 A IMPLY B 
                 pBW342 
                 25 
               
               
                   
                 B IMPLY A 
                 pBW343 
                 25 
               
               
                   
                 A NIMPLY B 
                 pBW344 
                 25 
               
               
                   
                 B NIMPLY A 
                 pBW345 
                 25 
               
               
                   
                 XOR 
                 pBW344 
                 12.5 
               
               
                   
                   
                 pBW345 
                 12.5 
               
               
                   
                 XNOR 
                 pBW334 
                 12.5 
               
               
                   
                   
                 pBW336 
                 12.5 
               
               
                   
                 TRUE 
                 pBW361 
                 25 
               
               
                   
                 FALSE 
                 pBW363 
                 25 
               
               
                   
                 XOR 
                 pBW448 
                 25 
               
               
                   
                 XNOR 
                 pBW450 
                 25 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 46 
               
             
            
               
                   
               
               
                 Transient transfection setup for 2-input, 4-output decoder  
               
               
                 circuit detailed in FIG. 2. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Marker, ng 
               
               
                 Reporter 
                   
                 Enzyme  
                 Enzyme  
                 Blank, ng 
                 pCAG- 
               
               
                 pEXPRCAG- 
                 Input 
                 1, ng 
                 2, ng 
                 pCAG- 
                 Lss- 
               
               
                 DECODER 
                 State 
                 pCAG-iCre 
                 pCAG-FlpO 
                 FALSE 
                 mOrange 
               
               
                 (pBW842) 
                 A B 
                 (pBW390) 
                 (pBW391) 
                 (pBW363) 
                 (pBW474) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 62.5 
                 0 0 
                 0 
                 0 
                 125 
                 62.5 
               
               
                   
                 1 0 
                 62.5 
                 0 
                 62.5 
                 62.5 
               
               
                   
                 0 1 
                 0 
                 62.5 
                 62.5 
                 62.5 
               
               
                   
                 1 1 
                 62.5 
                 62.5 
                 0 
                 62.5 
               
               
                   
               
            
           
         
       
     
     Tables 47A-47B: Transient transfection setup for 3-input logic gates detailed in  FIG. 3 . 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 47A 
               
               
                   
               
               
                   
                   
                 Enzyme 1, ng 
                 Enzyme 2, ng 
                 Enzyme 3, ng 
                 Blank, ng 
                 Marker, ng 
               
               
                   
                 Input 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
               
               
                   
                 State 
                 iCre 
                 FlpO 
                 VCre 
                 FALSE 
                 tagBFP 
               
               
                 Reporter 
                 A B C 
                 (pBW390) 
                 (pBW391) 
                 (pBW433) 
                 (pBW363) 
                 (pBW462) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Values 
                 0 0 0 
                 0 
                 0 
                 0 
                 0 
                 62.5 
               
               
                 Shown 
                 1 0 0 
                 41.7 
                 0 
                 0 
                 83.3 
                 62.5 
               
               
                 below 
                 0 1 0 
                 0 
                 41.7 
                 0 
                 83.3 
                 62.5 
               
               
                   
                 0 0 1 
                 0 
                 0 
                 41.7 
                 83.3 
                 62.5 
               
               
                   
                 1 1 0 
                 41.7 
                 41.7 
                 0 
                 41.7 
                 62.5 
               
               
                   
                 1 0 1 
                 41.7 
                 0 
                 41.7 
                 41.7 
                 62.5 
               
               
                   
                 0 1 1 
                 0 
                 41.7 
                 41.7 
                 41.7 
                 62.5 
               
               
                   
                 1 1 1 
                 41.7 
                 41.7 
                 41.7 
                 0 
                 62.5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 47B 
               
             
            
               
                   
               
               
                 Logic gate 
               
            
           
           
               
               
               
               
            
               
                   
                 Gate 
                 Plasmid ID 
                 ng 
               
               
                   
               
               
                   
                 FULL ADDER 
                 pBW820 
                 62.5 
               
               
                   
                 FULL SUBTRACTOR 
                 pBW840 
                 62.5 
               
               
                   
                 HALF ADDER-SUBTRACTOR 
                 pBW841 
                 62.5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 48 
               
             
            
               
                   
               
               
                 Transient transfection setup for six-input Boolean logic Look-up (LUT) Table genetic device. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 OOLEAN 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 LOGIC LUT, ng 
                   
                 Enzyme 1, ng 
                 Enzyme 2, ng 
                 Enzyme 3, ng 
                 Enzyme 4, ng 
                 Enzyme 5, ng 
                 Enzyme 6, ng 
                 Blank, ng 
                 Marker, ng 
               
               
                 PEXPRCAG- 
                 Input 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
                 pCAG- 
               
               
                 LUT 
                 State 
                 iCre 
                 FlpO 
                 PhiC31 
                 Vika 
                 B3 
                 bxb1 
                 FALSE 
                 tagBFP 
               
               
                 pBW829) 
                 A B 
                 (pBW390) 
                 (pBW391) 
                 (pBW440) 
                 (pBW434) 
                 (pBW435) 
                 (pBW439) 
                 (pBW363) 
                 (pBW462) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 75 
                 0 0 
                 0 
                 0 
                 0 or 25 
                 0 or 25 
                 0 or 25 
                 0 or 25 
                 Fill to 
                 25 
               
               
                   
                 1 0 
                 25 
                 0 
                   
                   
                   
                   
                 total DNA 
                   
               
               
                   
                 0 1 
                 0 
                 25 
                   
                   
                   
                   
                 amount = 
                   
               
               
                   
                 1 1 
                 25 
                 25 
                   
                   
                   
                   
                 250 ng 
               
               
                   
               
            
           
         
       
     
     Example 6 
     The switches are designed using DNA recombinase systems. DNA recombinases are enzymes derived from bacteria and fungi that recognize pairs of specific DNA sequences that are 30-40 base pairs long. The recombinase can perform either an excision or inversion on the sequence between these recognition sites based on the relative orientation of these sequences. Cre is a recombinase that acts upon pairs of sites called lox sites. A version of the stable inversion switch has also been constructed that responds to activity of the Flp recombinase, which acts on frt recognition sites. 
     There are several parameters that can guide the design of these switches. (1) Drug inducible: the switches can be controlled at bedside using the transient addition of a drug so that a doctor can easily change the therapy with minimal stress to the patient. (2) Stable switching: T cells undergo rapid proliferation upon activation, so it is important that the switches provide stable memory of switching from one state to another even under conditions of rapid expansion. (3) Lentiviral compatibility. 
     The stable inversion switch allows for control over whether a cell should stay in some initial state (State 1) or to switch to another state (State 2). These states can be encoded by different genes or promoters. The control over switching from State 1 to State 2 is effected by recombinase activity on the switch. We have constructed several variants of this switch ( FIG. 18 ): 1.) Target switch: Directs T-cell to a new target. 2.) Affinity switch: Tunes the dynamics of T-cell activation by changing between CARS with different affinity to same target. 3.) Expression level switch: Tunes dynamics of T-cell activity by changing expression level of CAR. 4.) “On” or “Off” switch: Controls when the therapy is active. 
     As part of the design, drug-inducible recombinases can be implemented, which allow one to control recombinase activity through drug addition. There are several drugs available for use to control the action of recombinases, including doxycycline, tamoxifen, and abscisic acid. These drugs can control recombinase activity through different mechanisms: transcriptional control (doxycycline, abscisic acid, rapamycin), dimerization (rapamycin), and nuclear localization (tamoxifen). With the exception of rapamycin-induced dimerization, which has only been developed for Cre, all of these mechanisms can be used to control both Cre and Hp activity. 
     Using the drug-inducible recombinases to act on the stable inversion designs described above, doctors will be allowed greater control over adoptive T-cell therapy. With these circuits, the therapy can be tuned to an individual patient&#39;s needs. For example, patient can begin the therapy in its safest iteration. The doctor can then evaluate the patient&#39;s response and use a drug to drive recombination and advance the T-cells to a more aggressive form of therapy ( FIG. 19 ). 
     In preliminary tests using a lox-based expression level switch to control mCherry expression human embryonic kidney (HEK) cells, expression of mCherry was increased by 10-fold through Cre activity upon the switch ( FIG. 20 ), Thus this design can be to create a stable change in expression of a gene, and we will use it to control CAR expression. 
     To create T-cells that express our circuit components, lentiviral integration can be used to stably integrate the switch and inducible recombinases into the genome. This lentiviral integration has been used successfully to express a fluorescent reporter stable inversion switch and a tamoxifen-inducible Cre in Jurkat T-cells. The reporter switch causes a cell to switch from mCherry expression to GET expression, and one were then able to induce this switch ( FIGS. 21A-21C ), These results indicate a drug can be used to control recombinase activity upon the stable inversion switch design. 
     A Jurkat line that expresses GFP under an NFAT promoter has also been used to monitor activation. With this line, the inventors have stably expressed the tamoxifen-inducible Cre and an “ON” switch that turns on expression of a Her2-specific CAR, Using this switch, the inventors were able to control the response of the T-cell to Her2 through the addition of tamoxifen ( FIGS. 22A-22C ). 
     These switches can be implemented in a patient&#39;s own T-cells. Constitutively active Cre and Flp are used to act on the fluorescent reporter stable inversion switch in CD4 primary T-cells. 
     Testing switches in HEK cell. 
     To track the efficacy and dynamics of the switches, fluorescent-tagged recombinases and CARs can be used in testing the switches. This tagging can allow one to use fluorescence-activated cell sorting (FACS) to track the expression of the switch components. The switches can be tested first in human embryonic kidney (HEK) cells due to the ease of transfection, which will allow for quick verification that all components function in mammalian cells. This testing can also involve the construction of a simple fluorescence circuit that expresses red fluorescence until recombinase activity causes the red fluorescence to turn off and green fluorescence to turn on. The red and green fluorescence provides an easy read-out that can provide one with information of how different inducers effect recombinase activity. 
     Constructing and Testing Stable Circuit Lines in Jurkat T-Cells. 
     As the goal is to be able to use these switches in T-cells, lentiviral integration can be used to create lines of Jurkat T-cells that stably express the switches and inducible recombinase components. The fluorescent switch is first tested with inducible recombinases to characterize recombinase activity and refine the induction protocol. There are several aspects of inducible recombination activity that these stable lines can help to characterize. (I) Basal activity: While the recombinases should only act when the designated inducer is added to the media, the level of control may not always be tight enough to restrict recombinases from acting when there is no inducer present. This leakiness can lead to leaky basal recombination activity. It is important to minimize this basal activity so that the switches can be used safely. (II) inducibility: While keeping the basal activity low is important, it is also important that upon drug induction, the recombinase is able to work efficiently to convert the cell to the desired state. 
     Different parameters of the protocol for inducible recombinase activity can be varied to determine the best method to achieve a safe and effective treatment, using the basal activity and inducibility to characterize the outcome. These parameters include the volume of inducible recombinase lentivirus used to transduce cells, the drug dosage used to induce recombinase activity, and the amount of time the cells are exposed to the drug. 
     To create stable lines expressing our CAR-expressing switches, a Jurkat line that expresses a GFP reporter under the NEAT promoter can be used, NFAT turns on during T-cell activation, so this line can be used to track Jurkat activation by measuring GFP expression. Plate-bound antigen as well as artificial antigen presenting cells can be used to activate the T-cell. The fluorescent-tagged recombinases and CARs can then be used to monitor induction and switching from State 1 to State 2, and the NEAT-GFP reporter to monitor T-cell activation. This tracking can allow one to characterize the dynamics of switching, and correlate these changes in CAR expression to the activity of the T-cell, which can allow one to explore questions like how long it takes to switch from State 1 to State 2, how different parameters of CAR expression affect activation, and how the presence of different CARs on one T-cell affects overall response. 
     Constructing and Testing Stable Circuit Lines in Primary T-Cells. 
     Lentiviral integration can be used to express our circuits in primary T-cells, using fluorescent read-outs to track the switching of the cells from State 1 to State 2. The response of the primary T-cells to the target antigen can be measured by measuring proliferation, interleukin-2 (IL2), and Interferon gamma (IFN-γ), and by monitoring the ability of the primary T-cells to kill artificial antigen presenting cells. 
     Testing Switches in Mice. 
     A Mouse xenograft tumor model can be developed using 6-8 week old female NOD/Scid mice. They are maintained under pathogen-free conditions. Ten female mice are subcutaneously inoculated on the flank with cells from cancer cell lines expressing different levels of antigen, and the T-cells will be added via retraorbital injection. To induce switching, drugs can be added to the food. Tumor dimensions can be measured using calipers, and the animals can be euthanized if the tumor reaches 2 cm in diameter. 
     A transduction and induction strategy can be designed that minimizes the toxicity of the recombinases while still maintaining their activity. These strategies can include expressing the inducible recombinase at low levels, tagging the recombinases with degradation tags, or introducing the inducer in pulses so that the recombinase activity is induced for only short periods of time. These strategies are oriented around lowering the amount of recombinase present in the system or limiting their exposure to the DNA. While immunogenicity and genotoxicity are important challenges to consider in our design, they are only toxic to the cells the circuit is expressed in. While this will reduce the efficacy of the therapy, it should not kill other cells. 
     Packaging. The size of the circuits described is limited by the amount of DNA that can be successfully packaged into a lentiviral, and transduction of switches decrease as they become larger. Several strategies can be explored to overcome the challenge of limited payload delivery. For switches like the affinity switch, where the circuit is designed to switch from the expression of one CAR to another, one can split the design into two separate viruses: one that encodes the first CAR in an “OFF” switch and another that encodes the second CAR in an “ON switch”. DNA transposon systems like PiggyBac and Sleeping Beauty can also be used to integrate the switches into T-cells. PiggyBac has been used to integrate CARs into T-cells. 
     With these components in place, the switches described herein can allow for increased control over the activity of CARs in T-cells, enabling adoptive T-cell therapy to become more personalized to an individual patient&#39;s needs. 
     Example 7 
     An ON/OFF toggle switch has been developed that can stably switch to either the ON or OFF state with the transient addition of drugs. This improvement can simplify treatment and avoid the potential long term effect of the drug on the patient. A quick OFF switch, an ON/OFF toggle switch, and a kill switch can all be implemented together to afford high level of control and safety. 
     To construct the ON/OFF toggle switch, several serine recombinases and corresponding recombinase directionality factors (RDFs, e.g., gp47 for bxb1, gp3 for phiC, ORF7 for TP901-1, gp25 for TG1, and gp3 for PhiRv1) that perform stable and reversible site specific DNA recombination were first used. Some of these enzymes (e.g., PhiC31 or bxb1) had been shown to be efficient in performing recombination reactions in mammalian cells. When the recombination sites, attB and attP are placed in the antiparallel orientation, the presence of recombinases will stably invert the DNA sequence between the two sites and generate an attL and attR site (“BP reaction”). This inversion remains stable unless a RDF is also expressed along with bxb1 or phiC, which will invert the sequence between attL and attR and regenerate attB and attP site (“LR reaction”). This reversible reaction has been demonstrated in  E. coli  to be repeatable for many cycles. 
     Experiments were performed to verify whether these recombinases with RDF can invert the sequence between attL and attR and regenerate attB and attP site (LR reaction) as well as BP reaction because not all recombinases and RDFs had been tested in mammalian cells to achieve 13P &amp; LR reaction. It turned out that every recombinase including PhiC31, TP901-1, TG1, and PhiRV1 can successfully undergo BP &amp; LR reaction. Among the recombinases and RDFs that can undergo BP and LR reaction, several inducible systems were screened for (e.g., ERT2, Destabilizing Domain (DD) from FKBP, DD from dihydrofolate reductase (DHFR), doxycycline inducible promoter) that can induce either recombinases ear RDFs and achieve BP &amp; LR reaction successfully. For recombinases (PhiC31, TP901-1, and TG1), both destabilizing domains (one from FKBP and the other from dihydrofolate reductase) worked well in inducing expression of recombinases, but did not work well in inducing RDFs. However, PhiRv1 did not get induced with two DD domains efficiently. Also, location of destabilizing domain matters. When DD was fused to the C-terminus, it worked better than when it was fused to the N-terminus. When DD was fused to both the N and C terminus, low basal expression was observed. In addition, DD from DHFR (up to 8˜9 fold difference) worked better than DD from FKBP (up to 3˜4 fold difference). Shield1 ligand was used to induce DD from FKBP domain and trimethoprim (TMP) was used to induce DD from dihydrofolate reductase). Doxycycline inducible promoter (both Tet-on system and Tet-on-3G) system was used to induce recombinases and RDFs, but doxycycline inducible promoter was not efficient at expressing recombinases and RDFs. Also, ERT2 domain which is induced by 4-hydrotamoxifen (40HT) was used. ERT2 was primarily tested to induce different RDFs (e.g., gp3, gp25, and ORF7) and ERT2 can be used to induce gp3, gp25 and ORF7. Location of ERT2 domain matters. Most of the time when ERT2 was fused both the N and C terminus, basal expression was low and gave high fold change (˜8). Location of NLS can be changed (either the Nor C terminus or both) to give better expression level. 
     The inventors have demonstrated the use of three widely used recombinases and corresponding RDFs (parenthesis), PhiC31 (gp3), TG1 (gp25) and TP901-1 (ORF7) in mammalian cells. These recombinases and corresponding RDFs were induced by different drugs (Shield1, TMP, and 40HT) to make On/Off toggle switch. Recombinases (PhiC13, TG1, and TP901-1) were induced by (Shield1, and TMP) and corresponding RDFs were induced by 40HT. 
     EQUIVALENTS 
     While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. 
     All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. 
     The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” 
     The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. 
     As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another 
     embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. 
     It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. 
     In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. 
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