# Patent

## Title
Method for increasing protein expression in cells

## Abstract

The present invention relates to a method for increasing the expression of a protein in cells, preferably in eukaryotic cells, by reducing the number of RNase L cleavage sites in the coding and/or non-coding region of the nucleic acid sequence of said protein. Furthermore, it relates to nucleic acid sequences exhibiting a reduced number of RNase L cleavage sites as well as to the proteins translated from such sequences.

## Background
CROSS REFERENCE TO A RELATED APPLICATION
This application is a National Stage Application of International Application Number PCT/EP2010/000271, filed Jan. 19, 2010; which claims priority to International Application PCT/EP2009/000302, filed on Jan. 19, 2009; all of which are incorporated herein by reference in their entirety.
The Sequence Listing for this application is labeled “As-filed_ST25.txt”, which was created on Jul. 14, 2011, and is 40 KB. The entire contents are incorporated herein by reference in their entirety.
The present invention relates to a method for increasing the expression of a protein in cells, preferably in eukaryotic cells by reducing the number of RNase L cleavage sites in the coding and/or non-coding region of the nucleic acid sequence of said protein. Furthermore, it relates to nucleic acid sequences exhibiting a reduced number of RNase L cleavage sites as well as to the proteins translated from such sequences.
Transient response genes regulate critical biological responses that include cell proliferation, signal transduction events, and responses to exogenous agents, such as inflammatory stimuli, microbes, and radiation (Lai et al., 2006; Lal et al., 2004; Lopez de Silanes et al., 2005). They are controlled by both cis-acting factors such as specific sequence elements and trans-acting factors like certain RNA binding proteins. Sequence elements, mainly in the 3′ untranslated region (3′UTR) and modulation by RNA binding proteins can affect messenger RNA (mRNA) stability and protein translation.
An appreciable number of genes harbor destabilizing sequence elements in the 3′ UTR of their mRNA, mostly adenylate-uridylate (AU)-rich elements (AREs). These AREs comprise a heterogeneous group of sequence classes that can affect protein interactions with the mRNA, and therefore influence the mRNA decay characteristics (Bakheet et al., 2006; Barreau et al., 2005). The stabilization of cellular mRNAs can occur by the activity of mRNA stabilization-promoting proteins, such as the HuR protein, or by inactivation of RNA decay promoting proteins, such as the zinc finger protein tristetraprolin (TTP).
A different class of trans-acting factors that can affect cellular mRNAs is the endoribonuclease Ribonuclease L (RNase L), which is an ubiquitous intracellular enzyme that has previously been thought to be specific to viral mRNAs. However, recent studies showed that RNase L can also participate in the transient response of certain biological processes (Khabar et al., 2003a; Li et al., 2000). RNase L is considered to be a part of the interferon (IFN) system. IFN induces gene expression of the enzyme oligoadenylate synthetase (OAS) which, upon binding to viral double-stranded RNA intermediates, becomes activated and synthesizes short 2′-5′oligoadenylates (2-5A). These, in turn, activate RNase L, which potently degrades viral mRNAs. RNase L is activated by subnanomolar levels of 2-5A, resulting in the cleavage of single-stranded regions of viral RNA, preferentially after UU and UA dinucleotides in viral mRNAs (Wrechester et al., 1981; Han et al., 2004). At higher levels, RNase L may lead to broader effects such as cleavage of 18 S and 28 S ribosomal RNAs (Wreschner et al., 1981).
In recent years it has become widely accepted that RNase L participates in the degradation of selected cellular mRNAs (Bisbal et al., 2000; Chandrasekaran et al., 2004; Khabar et al., 2003b; Le Roy et al., 2001; Li et al., 2000). Specifically, RNase L has been shown to down-regulate PKR mRNA (Khabar et al., 2003b). During the IFN antiviral response in normal cells, PKR mRNA expression is transient, but in RNase L-null cells, extended kinetics of PKR mRNA expression is observed due to increased mRNA stability (Khabar et al., 2003b). The effect results in prolongation of the PKR-dependent phosphorylation of the subunit of eukaryotic translation initiation factor 2, eIF2α, a process that leads to inhibition of viral protein synthesis (Khabar et al., 2003b). Thus, RNase L contributes to the transient nature of the IFN response in order to ensure a brief translational arrest imparted by PKR. A similar role of RNase L negative regulation of the IFN response has also been suggested by the report that a novel IFN-stimulated gene encoding a 43-kDa ubiquitin-specific protease, designated ISG43, is down-regulated by RNase L (Li et al., 2000). This regulation occurs at the level of mRNA stability, since the ISG43 mRNA half life increases in RNase L-null cells (Li et al., 2000). RNase L can down-regulate another functionally important cellular mRNA, myoD, encoding an import transcription factor essential for muscle differentiation. RNase L and its inhibitor RLI are sequentially induced during C2 cell line myoblast differentiation to myotubes (Bisbal et al., 2000). Inhibition or over-expression of RNase L prolongs or decreases MyoD mRNA half life, respectively (Bisbal et al., 2000). Since a pool of RNase L molecules localizes to mitochondria and is increased following IFN-α treatment, a role of RNase L in down-regulating mitochondrial mRNAs, such as those of CYTB, ATPase 6 (ATP6), and cytochrome oxidase II (CO), has been proposed as a mechanism of the anti-proliferative action of IFN (Le Roy et al., 2001). This was demonstrated by reducing RNase L activity through the introduction of an antisense construct or by directly activating RNase L activity by 2-5A (Le Roy et al., 2001). It has further been shown that RNase L exhibits a preference for viral mRNA, for example encephalomyocarditits virus (EMCV), when compared to non-viral mRNAs, particularly in conditions where the levels of 2-5A is limiting. (Li et al., 1998a). The effects of RNase L on cellular mRNAs appear to be highly restricted to specific mRNAs, since no global effects on cellular mRNAs are observed in the studies that have dealt with this topic. Furthermore, none of the above studies demonstrates a direct binding of RNase L to cellular target mRNAs or a sequence specificity as shown for viral mRNAs. Thus, the mechanism of RNase L activity in connection with non-viral cellular mRNAs remains largely unclear.
Dominant negative forms of RNase L have previously been generated by either amino acid substitutions or by truncation of the full length protein. For example, a dominant negative RNase L, ZB1, inhibits the antiviral and anti-proliferative action of wild type RNase L; it is a truncated form of murine RNase L, which lacks 89 carboxy-terminal amino acids (Hassel et al., 1993). Dong et al., 2001 describe other truncations and point mutations of RNase L, e.g. mutations in the nuclease domain (R667A).
U.S. Pat. No. 6,762,038 suggests the use of mutant embryonic fibroblasts cell lines (MEFs) generated from mice having a homozygous disruption in their RNase L gene (Zhou et al., 1997) for enhanced expression of transfected genes. However, the effect of enhanced expression is restricted to these particular cell lines. Furthermore, the creation of new RNase L-null cell lines is a very laborious process and not readily applicable to all mammalian and/or eukaryotic expression systems.
Mammalian expression systems have become an important means for therapeutic protein and antibody production and possess several advantages over prokaryotic expression systems, e.g. with respect to proper protein folding and posttranslational modifications, such as glycolysation. However, in many systems, low protein expression yields represent a costly technical obstacle. This is particularly problematic, if the desired protein is inherently difficult to express, e.g. membrane proteins, such as G-protein linked receptors (GPCRs), large proteins, antibodies, fusion proteins, protein complexes, vaccines, and blood plasma proteins. Reasons for this difficulty could be an inherent instability of the protein itself, an inherent instability of the mRNA, as it is the case for AU-rich elements containing mRNAs, or weak promoter activity.
Common solutions to these problems focused on increasing the expression of proteins by providing strong promoters, such as the CMV promoter, and enhancer elements upstream or downstream of the promoter including specific types of introns, such as intron A of CMV. Other solutions involved chromatin Matrix Attachment Region (MAR) elements which are 300-3000 base pairs long DNA elements that are important in nuclear and chromosomal architecture. It was proposed that these elements prevent the neighbouring chromatin from affecting transgene expression, which leads to an increased probability of isolating a clone exhibiting the desired regulated expression (U.S. Pat. No. 7,129,062). This particular approach is potentially problematic as it may involve several different vector constructs to achieve the effect. Despite the availability of these approaches, there is still a need to further increase the protein expression of proteins that are difficult to express, such as those mentioned above.
Codon optimization (or codon usage optimization) is another method known in the art to boost protein production. It is based on the observation that, if a nucleic acid sequence encoding the protein to be expressed contains codons (triplets) that are rarely used by the host, its expression level will not be maximal. Codon optimization basically involves altering the rare codons in the target nucleic acid sequence, so that they more closely reflect the codon usage of the host. The information usually used for the optimization process is therefore the DNA or protein sequence to be optimized and a codon usage table of the respective host (see for example Table 1 for the human genome). The codon usage table lists the relative frequency of each possible codon for a particular amino acid in a given organism. A full list of codon usage bias in all organisms is found on the website: http://www.kazusa.or.jp/codon/. Several web-based programs are also available to optimize codons based on codon usage bias for a given host organism. Codon optimization may be successful in some situations where genes of non-human or non-mammalian origin are expressed in human or other mammalian host cells, and vice versa. However, codon usage is just one of many factors influencing the expression level of a protein, and the effect of codon optimization is often limited.
It was an object of the present invention to provide for a method to significantly improve the yield of endogenous and exogenous (recombinant) proteins expressed in cells of any organism, preferably in eukaryotic cells, including proteins that are inherently difficult to express. This method should be time and cost efficient and should allow for the large-scale production of proteins in cells of any type of organism including prokaryotic and eukaryotic cells. It was another object of the present invention to achieve this improvement in expression yields without changing regulatory approved features for the production of recombinant proteins, such as the cell lines used, recombinant protein characteristics, or the use of exogenous materials, in order to allow for the production of therapeutically used proteins.
The objects of the present invention are solved by a method for increasing the expression of a protein in cells, preferably eukaryotic cells, said method comprising the step of reducing the number of RNase L cleavage sites in the nucleic acid sequence of said protein. In one embodiment, said cells are prokaryotic cells; in another embodiment, said cells are eukaryotic cells.
The nucleic acid sequence of a protein comprises both coding and non-coding regions, i.e. regions that are translated into a sequence of amino acids (also referred to as exons) and regions that are not translated into a sequence of amino acids. Non-coding regions of the nucleic acid sequence are for example the 5′ untranslated region (5′UTR), the 3′ untranslated region (3′UTR), and introns. All of these elements (5′UTR, 3′UTR, introns, and the coding region) can control gene and protein expression, and are, thus, targets for the above method. According to the invention, said step of reducing the number of RNase L cleavage sites reduces said number either in the coding region or non-coding region, or in both.
In one embodiment said number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50% (compared to the number of RNase L cleavage sites in the wild type nucleic acid sequence).
Preferably said cleavage sites are UU and/or UA dinucleotides.
RNase L is an endoribonuclease, and is, thus, only active on the RNA level (both primary RNA, i.e. unspliced, and mRNA, i.e. spliced). UU and UA dinucleotides only occur on the RNA level.
However, it is preferred that said step of reducing the number of RNase L cleavage sites in the nucleic acid sequence of said protein is performed on the DNA level: UU and UA dinucleotides in an RNA sequence correspond to TT and TA dinucleotides in a DNA sequence. Techniques that allow to specifically change a given DNA sequence are well know in the art and include, but are not limited to gene synthesis, site-directed mutagenesis, deletion mutations by restriction digestion, and mutation introduction by recombination. The technique of gene synthesis is particularly preferred according to the present invention.
In one embodiment of the present invention said step of reducing the number of RNase L cleavage sites reduces said number in the coding region of said nucleic acid sequence.
Preferably said step of reducing the number of RNase L cleavage sites in said nucleic acid sequence is performed without altering the amino acid sequence of said protein. The open reading frame (ORF) is, thus, not altered by said step.
In one embodiment in said step of reducing the number of RNase L cleavage sites a codon comprising a UU and/or UA dinucleotide is exchanged for an alternative codon not comprising a UU and/or UA dinucleotide and coding for the same amino acid.
In one embodiment in said step of reducing the number of RNase L cleavage sites at least one codon of an adjacent pair of codons comprising a UU and/or UA dinucleotide is exchanged for an alternative codon coding for the same amino acid so that said adjacent pair of codons does no longer comprise a UU and/or UA dinucleotide.
Preferably the first codon of said adjacent pair of codons comprising a UU and/or UA dinucleotide is exchanged.
In one embodiment said alternative codon is the more frequently used codon in said cells, preferably in said eukaryotic cells.
In one embodiment of the present invention said step of reducing the number of RNase L cleavage sites reduces said number in the non-coding region of said nucleic acid sequence.
Preferably said non-coding region is a 5′UTR, a 3′UTR, or an intron.
Examples for introns include, but are not limited to the CMV intron, SV40 intron, rabbit beta globin intron (RBTG), and synthetic introns.
In one embodiment said step of reducing the number of RNase L cleavage sites in the non-coding region of said nucleic acid sequence is performed by mutation, deletion, or insertion of nucleotides.
Preferably said step of reducing the number of RNase L cleavage sites in the non-coding region of said nucleic acid sequence does not alter functionally important elements in the non-coding region, such as sequences in the 5′UTR that are close to the initiation codon (ATG), since they may harbor translation enhancing sequences (e.g. kozac), the poly A signal in the 3′UTR (e.g. AAUAAA or AUUAAA) or other necessary or accessory sequence elements used for polyadenylation, intron-exon junctions/boundaries, splicing branch points and exon donor/acceptor splice sites in introns, and the CT-rich area between the splice acceptor site to the end of the branch point. For example there is an U-rich 50 nucleotide region that is downstream of the strong poly A signal, which should not be altered when possible (Legendre and Gautheret, 2003).
Preferably said step of reducing the number of RNase L cleavage sites in the non-coding region does not

    
    
        (a) change the GC content of an intron to more than 80% and its length to less than 80%,
        (b) change the GC content of a 5′UTR to more than 80%, and
        (c) change the GC content of a 3′UTR to more than 80% and its length to less than 80%.
    
    


In one embodiment above method further comprises the step of codon optimization prior to said step of reducing the number of RNase L cleavage sites.
In one embodiment above method further comprises the step of transfecting said nucleic acid sequence of said protein into said cells, preferably into said eukaryotic cells in form of an expression active PCR product or contained in an expression vector after said step of reducing the number of RNase L cleavage sites.
The term “expression active PCR product” as used herein is meant to refer to a PCR product that is generated by PCR amplification using two primers complementary to sequences flanking the DNA sequence of interest, such as a cDNA, an open reading frame, or a gene that is contained in an expression vector, wherein the resulting PCR product contains a promoter, the DNA sequence of interest, and a termination sequence, and allows the expression of the DNA of interest, when transfected to a host cell (see also: Al-Zoghaibi et al., 2007).
According to the present invention any expression vector can be used, however, eukaryotic/mammalian expression vectors are preferable. Mammalian expression vectors are widespread tools to study the biological function of a protein, and various types (e.g. plasmid-based or viral-based vectors) are known in the art. Suitable expression vectors are not limited to a specific promoter, 5′UTR, 3′UTR, or intron. They can be constitutively expressed, inducible, repressed or regulatable.
According to the invention, it is preferred that the promoter is eukaryotic and the termination site is a poly A sequence containing a polyadenylation signal. Eukaryotic or mammalian promoters are known in the art and include, but are not limited to cytomegalovirus (CMV) immediate early promoter, SV40 promoter, elongation factor (EF) promoter, RSV promoter, and chicken β-actin promoter. Preferred eukaryotic polyadenylation signals are bovine growth factor (BGH) poly site, growth hormone poly A, SV40 poly A, and HSK poly A (Foecking and Hofstetter, 1986; Kobayashi et al., 1997). Examples of eukaryotic terminators are bovine growth factor (BGH) poly A site, SV40 poly A, HSK poly A, and synthetic poly A.
Methods for transiently or stably transfecting cells with DNA/vectors are well known in the art. These include, but are not limited to calcium phosphate co-precipitation, electroporation, cationic polymer transfection, and liposome-mediated transfection (e.g. lipofection). Reagents for liposome-mediated transfection are commercially available, e.g. lipofectamine (Invitrogen) and polyethylenimine (Sigma). Cells can also be transfected by viral transfection or via viral coat particles. Another preferred method for the transfection of cells according to the present invention is the in vivo microinjection of said expression active PCR product or said expression vector and selectively growing the cells containing said expression active PCR product or said expression vector with or without the help of a selection drug. Thus, the expression active PCR product or expression vector in accordance with the present invention may additionally comprise a selectable marker.
For the generation of stable cell lines, clones can be selected using various selectable markers, which include, but are not limited to neomycin, blasticidin, puromycin, zeocin, hygromycin, and dihydrofolate reductase (dhfr).
Suitable eukaryotic/mammalian cells (host cells) for all of the above methods are also well known in the art and include, but are not limited to CHO, HEK 293, HeLa, and COS-7 cells.
In one embodiment the above method further comprises the step of translating said protein from said expression active PCR product or expression vector in said cells, preferably in said eukaryotic cells.
Preferably said protein is selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.
The objects of the present invention are also solved by a nucleic acid sequence, wherein the number of RNase L cleavage sites is reduced by at least 10%, preferably at least 25%, more preferably at least 50% (compared to the number of RNase L cleavage sites in the wild type nucleic acid sequence).
Preferably said nucleic acid sequence is produced by the method as described above.
Preferably said nucleic acid sequence is the nucleic acid sequence of a protein selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.
In one embodiment said nucleic acid sequence has a sequence selected from SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 24, 26, 28, 30-36, preferably SEQ ID NO: 9, 15, 17, 23, 30-36.
The objects of the present invention are also solved by an expression active PCR product or an expression vector comprising the nucleic acid as described above.
The objects of the present invention are further solved by a host cell containing the above expression active PCR product or expression vector.
Finally, the objects of the present invention are also solved by a protein produced by the method as described above, wherein said protein is selected from the group comprising reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.
According to the present invention the term “therapeutic proteins” is meant to include proteins used in a pharmaceutical context, such as antibody fragments, immunoglobulin chains, heavy and light chains, Fab fragments, enzymes, growth factors, interferons, cytokines, lymphokines, adhesion molecules, receptors, as well as derivatives or fragments thereof.
According to the present invention reporter proteins (either alone or fused to another protein) are particularly preferred “targets” for the above described method. There are many fluorescent and non-fluorescent reporter proteins including (without being limited to) green fluorescent proteins (GFP), red fluorescence proteins (RFP), yellow fluorescent proteins (YFP), blue and cyanine fluorescent proteins (CFP), luciferase, secreted alkaline phosphatase (SEAP), Chloramphenicol acetyltransferase (CAT), secreted hormone, secreted cytokine, β-galactosidase, and other fluorescent and bioluminescent proteins. The choice of reporter protein depends on the cell line used (endogenous activity), the nature of the experiment (e.g. dynamics of gene expression and transfection efficiency), and the adaptability of the assay to the chosen detection method (Naylor 1999). Several modifications of the reporter proteins themselves have been sought to improve the reporter performance, such as rapid response and magnitude of change, e.g. the use of destabilization elements (Li et al., 1998b; Zhao et al., 1995). Green fluorescent protein (GFP) and other fluorescent proteins are increasingly popular for the non-invasive monitoring of gene expression in living tissues, cells, and in laboratory animals (Naylor 1999). Thus, GFP (and its derivatives, such as EGFP), other fluorescent proteins (see above), as well as fusion proteins comprising a fluorescent protein are even more preferred “target proteins” for the method according to the present invention.
The inventor has surprisingly found that reducing the number of RNase L cleavage sites, preferably UU and UA dinucleotides, in the nucleic acid sequence of a protein to be expressed in a eukaryotic and/or mammalian expression system results in a significantly improved expression and yield of said protein. Without wishing to be bound to a certain theory, it is believed that the reduction of RNase L cleavage sites (i.e. reduced frequency of these sites) results in a nucleic acid sequence, namely an RNA—both primary RNA and mRNA—, that is less prone (i.e. more resistant) to attacks of endogenous RNase L in the host cells, preferably in the eukaryotic host cells, leading to increased mRNA stability and, thus increased protein expression.
Because of the universal concept involved, the present invention can be applied to any sequence. More specifically, the method according to the present invention is applicable to both endogenous and exogenous (recombinant) proteins/genes, to both stably integrated and transiently expressed genes, to proteins/genes that are difficult to express, and also to proteins/genes that are endogenously expressed in low-abundance. It can be applied to both prokaryotic and eukaryotic systems. The approach described herein will lead to a significant reduction of time and costs spent on protein production, and, thus, will allow a more efficient production of proteins/genes used as biopharmaceuticals, such as erythropoietin, growth factors, interferons, insulin, therapeutic and diagnostic antibodies, and protein- or peptide-based vaccines. It is especially useful for the expression of genes/proteins that have been proven to be very difficult to express/produce in large quantities. Examples include, but are not limited to membrane proteins, such as G-protein linked receptors (GPCR), large proteins, antibodies, fusion proteins, protein complexes, vaccines, and blood plasma proteins.
It can also help to significantly improve the expression and performance of reporter proteins, e.g. fluorescent proteins, such as GFP or luciferase, and, thus, increase the sensitivity of methods using such reporter proteins (fluorescence or luminescence microscopy, fluorescence-based microarrays, cell-sorting, etc.).
Furthermore, the approach is particularly practical and simple, since it is applicable to any cell line, such as cell lines used in the biotechnology industry including (but not limited to) hamster CHO1 and HEK293.
The following describes general principles for the step of reducing the number of RNase L cleavage sites according to the present invention:
Reduction of the Number of UU and/or UA Dinucleotides in a Coding Region
Table 2 and Table 3 show the changes that can be made. It is important to note that these modifications are entirely different from the codon usage frequency tables (see for comparison Table 1) that are used to optimize codons for protein expression on the basis of their codon bias (codon usage frequency) in a given organism. The present method is not directed at changing codons on the basis of the codon usage frequency, but in order to reduce the number of RNase L cleavage sites/targets (UU and UA dinucleotides). Moreover, the method according to the present invention is not “organism-dependent”; the number/frequency of UU and/or UA dinucleotides can be reduced in any gene (nucleic acid sequence) from any organism, as long as the corresponding amino acid sequence remains unaltered (see Tables 2 and 3). Furthermore, the reduction of the number of UU and/or UA dinucleotides can also be combined with the classical codon usage optimization for the desired organism (see for example Table 1), possibly resulting in an even more increased expression.
Table 2 shows the codons that harbor UU and/or UA dinucleotides as well as their non-UU/UA-harboring alternative(s). UUU coding for phenylalanine (Phe) can only be changed to UUC, since there is not other codon for phenylalanine that is totally devoid of UU or UA. This is also the case for tyrosine (UAU) that can only be changed to UAC.
Once the changes according to Table 2 are performed, di-triplets that form a UU or UA, i.e. NNU UNN or NNU ANN (with N being any nucleotide) are changed according to Table 3. If there is more than one alternative, preference is given to the more frequently used codon in the respective organism or in highly expressed genes.
Steps for UU and/or UA dinucleotide reduction in coding regions:

    
    
        1. Change codons according to Table 2.
        2. If more than one alternative codon exist, use the more frequently used codon in the desired organism (optional).
        3. Change the first codon of the di-triplets that form a UU or UA dinucleotide together (NNU UNN, NNU ANN) according to Table 3.
        4. If more than one alternative codon exist, use the more frequently used codon or the strongest for expression in the desired organism (optional, see Tables 4 and 5).
        5. Classical codon usage optimization (based on the host cell organism or on a list of codons most frequently used in high expression genes, can be performed optionally and preferably prior to the UU and UA reduction.

Reduction of the Number of UU and/or UA Dinucleotides in an Intron

    
    


Steps for UU and/or UA dinucleotide reduction in introns:

    
    
        1. Mutate or delete one or two of the two nucleotides in UU/UA dinucleotides: UU or UA to UC, UG, GA, CA, or TA. Alternatively, insert one nucleotide.
        2. The entire GC content of the intron should not be more than 80% and the length should not be changed to less than 80% of its original length.
        3. Do not change exon-intron boundaries including exon donor and acceptor splice sites and branch points. Avoid disrupting the CT-rich area ranging from the splice acceptor site to the end of the branch point.

Reduction of the Number of UU and/or UA Dinucleotides in a 5′UTR

    
    


Steps for UU and/or UA dinucleotide reduction in 5′UTRs:

    
    
        1. Mutate UU or UA to UC, UG, GA, CA, or TA.
        2. The entire GC content of the 5′UTR should not be more than 80%.
        3. Avoid context sequences near the initiation codon, ATG, since they may harbor translation enhancing sequences, such as kozac.

Reduction of the Number of UU and/or UA Dinucleotides in a 3′UTR

    
    


Steps for UU and/UA dinucleotides reduction in 3′ UTRs:

    
    
        1. Mutate or delete one or two of the two nucleotides in UU/UA dinucleotides: UU or UA to UC, UG, GA, CA, or TA. Alternatively, insert one nucleotide.
        2. The entire GC content of the 3′UTR should not be more than 80% and the length should not be changed to less than 80% of its original length.
        3. Do not change polyA signals such as AAUAAA or AUUAAA and avoid to alter any necessary or accessory sequence elements used for polyadenylation, if found.

Possible Changes in Non-Coding Regions

        1. Mutation: NUUN or NUAN to NUSN (where S is G or C, and N is any nucleotide)
        2. Insertion: NUUN or NUAN to NUSUN or NUSA (where S is G or C, and N is any nucleotide)
        3. Deletion: NUUN to NUS (where S is G or C, and N is any nucleotide)
        4. Deletion: NUAN to NAN or NUS (where S is G or C, and N is any nucleotide)

Tables

    
    









TABLE 1





Codon frequency in human genes


fields: [triplet] [amino acid] [fraction] [frequency: per thousand] ([number])


















UUU F 0.46 17.6  (714298)
UCU S 0.19 15.2  (618711)
UAU Y 0.44 12.2 (495699)
UGU C 0.46 10.6 (430311)


UUC F 0.54 20.3  (824692)
UCC S 0.22 17.7  (718892)
UAC Y 0.56 15.3 (622407)
UGC C 0.54 12.6 (513028)


UUA L 0.08  7.7  (311881)
UCA S 0.15 12.2  (496448)
UAA * 0.30  1.0  (40285)
UGA * 0.47  1.6  (63237)


UUG L 0.13 12.9  (525688)
UCG S 0.05  4.4  (179419)
UAG * 0.24  0.8  (32109)
UGG W 1.00 13.2 (535595)


 


CUU L 0.13 13.2  (536515)
CCU P 0.29 17.5  (713233)
CAU H 0.42 10.9  (441711)
CGU R 0.08  4.5 (184609)


CUC L 0.20 19.6  (796638)
CCC P 0.32 19.8  (804620)
CAC H 0.58 15.1  (613713)
CGC R 0.18 10.4 (423516)


CUA L 0.07  7.2  (290751)
CCA P 0.28 16.9  (688038)
CAA Q 0.27 12.3  (501911)
CGA R 0.11  6.2 (250760)


CUG L 0.40 39.6 (1611801)
CCG P 0.11  6.9  (281570)
CAG Q 0.73 34.2 (1391973)
CGG R 0.20 11.4 (464485)


 


AUU I 0.36 16.0  (650473)
ACU T 0.25 13.1  (533609)
AAU N 0.47 17.0  (689701)
AGU S 0.15 12.1 (493429)


AUC I 0.47 20.8  (846466)
ACC T 0.36 18.9  (768147)
AAC N 0.53 19.1  (776603)
AGC S 0.24 19.5 (791383)


AUA I 0.17  7.5  (304565)
ACA T 0.28 15.1  (614523)
AAA K 0.43 24.4  (993621)
AGA R 0.21 12.2 (494682)


AUG M 1.00 22.0  (896005)
ACG T 0.11  6.1  (246105)
AAG K 0.57 31.9 (1295568)
AGG R 0.21 12.0 (486463)


 


GUU V 0.18 11.0  (448607)
GCU A 0.27 18.4  (750096)
GAU D 0.46 21.8  (885429)
GGU G 0.16 10.8 (437126)


GUC V 0.24 14.5  (588138)
GCC A 0.40 27.7 (1127679)
GAC D 0.54 25.1 (1020595)
GGC G 0.34 22.2 (903565)


GUA V 0.12  7.1  (287712)
GCA A 0.23 15.8  (643471)
GAA E 0.42 29.0 (1177632)
GGA G 0.25 16.5 (669873)


GUG V 0.46 28.1 (1143534)
GCG A 0.11  7.4  (299495)
GAG E 0.58 39.6 (1609975)
GGG G 0.25 16.5 (669768)





Source: http://www.kazusa.or.jp













TABLE 2







UU/UA-harboring codons and their


non-UU/UA-harboring alternative(s)











UU/UA-

Alternative



harboring codon
Amino acid
codon(s)







UUU
Phenylalanine (F)
UUC



 



UUA
Leucine (L)
CUC, CUG



 



UUG
Leucine (L)
CUC, CUG



 



CUU
Leucine (L)
CUC, CUG



 



CUA
Leucine (L)
CUC, CUG



 



UAU
Tyrosine (Y)
UAC



 



AUU
Isoleucine (I)
AUC



 



AUA
Isoleucine (I)
AUC



 



GUU
Valine (V)
GUC, GUG



 



GUA
Valine (V)
GUC, GUG

















TABLE 3







Di-Triplets forming UU/UA dinucleotides and the


corresponding UU/UA-reduced di-triplet(s)









UU/UA-




forming




ditriplets
Amino acid
Modified di-triplets





UCU UNN
Serine (S) UNN
AGC UNN


 


UCU ANN
Serine (S) ANN
AGC ANN


 


CCU UNN
Proline (P) UNN
CCC UNN or CCA UNN


 


CCU ANN
Proline (P) ANN
CCC ANN or CCA ANN


 


ACU UNN
Threonine (T) 
ACC UNN or ACA UNN or



UNN
ACG UNN


 


ACU ANN
Threonine (T) 
ACC ANN or ACA ANN or



ANN
ACG ANN


 


GCU UNN
Alanine (A) 
GCC UNN or GCA UNN



UNN
GCG UNN


 


GCU ANN
Alanine (A) 
GCC ANN or GCA ANN



UNN
GCG ANN


 


CAU UNN
Histidine (H) 
CAC UNN



UNN



 


CAU ANN
Histidine (H) 
CAC ANN



ANN



 


AAU UNN
Asparagine (N) 
AAC UNN



UNN



 


AAU ANN
Asparagine (N) 
AAC ANN



ANN



 


GAU UNN
Aspartic acid
GAC UNN



(D) UNN



 


GAU ANN
Aspartic acid
GAC ANN



(D) ANN



 


UGU UNN
Cysteine (C) UNN
UGC UNN


 


UGU ANN
Cysteine (C) ANN
UGC ANN


 


CGU UNN
Arginine (R) UNN
CGC UNN or CGA UNN or




AGA UNN or AGG UNN


 


CGU ANN
Arginine (R) ANN
CGC ANN or CGA ANN or




AGA ANN or AGG ANN


 


AGU UNN
Serine (S) UNN
AGC UNN or UCC UNN or




UCA UNN or UCG UNN


 


AGU ANN
Serine (S) ANN
AGC ANN or UCC ANN or




UCA ANN or UCG ANN


 


GGU UNN
Glycine (G) UNN
GGA UNN or GGC UNN or




GGG UNN


 


GGU ANN
Glycine (G) UNN
GGA ANN or GGC ANN or




GGG ANN





UNN: UUC, UCU, UCC, UCA, UCG,


ANN: AUC, AUG, ACU, ACC, ACA, ACG, AAU, AAC, AAA, AAG, AGU, AGC, AGA, AGG













TABLE 4







Combination of UU/UA dinucleotide reduction


and classical codon optimization


(for single codons/triplets)












Changed
Codon


UU/UA codon
Amino Acid
Codons
bias*





UUU
Phenylalanine (F)
UUC
none


 


UUA
Leucine (L)
CUC, CUG
CUG


 


UUG
Leucine (L)
CUC, CUG
CUG


 


CUU
Leucine (L)
CUC, CUG
CUG


 


CUA
Leucine (L)
CUC, CUG
CUG


 


UAU
Tyrosine (Y)
UAC
None


 


AUU
Isoleucine (I)
AUC
None


 


AUA
Isoleucine (I)
AUC
None


 


GUU
Valine (V)
CUC, GUG
GUG


 


GUA
Valine (V)
CUC, GUG
GUG





*Human codon bias as an example of organism codon bias.













TABLE 5







Combination of UU/UA dinucleotide reduction and classical codon optimization


(for codon pairs/di-triplets)










WU Di-


Codon


Triplets
Amino Acid
Modified Di-Triplets
Bias*





UCU UNN
Serine (S) UNN
AGC UNN or TCG UNN or TCC UNN
AGC UNN


 


UCU ANN
Serine (S) ANN
AGC ANN or TCG UNN or TCC ANN
AGC UNN


 


CCU UNN
Proline (P) UNN
CCC UNN or CCA UNN
CCC UNN


 


CCU ANN
Proline (P) ANN
CCC ANN or CCA ANN
CCC ANN


 


ACU UNN
Threonine (T) UNN
ACC UNN or ACA UNN or ACG UNN
ACC UNN


 


ACU ANN
Threonine (T) ANN
ACC ANN or ACA ANN or ACG ANN
ACC ANN


 


GCU UNN
Alanine (A) UNN
GCC UNN or GCA UNN GCG UNN
GCC UNN


 


GCU ANN
Alanine (A) ANN
GCC ANN or GCA ANN GCG ANN
GCC ANN


 


CAU UNN
Histidine (H) UNN
CAC UNN
None


 


CAU ANN
Histidine (H) ANN
CAC ANN
None


 


AAU UNN
Asparagine (N) UNN
AAC UNN
none


 


AAU ANN
Asparagine (N) ANN
AAC ANN
none


 


GAU UNN
Aspartic acid (D) UNN
GAC UNN
none


 


GAU ANN
Aspartic acid (D) ANN
GAC ANN
none


 


UGU UNN
Cysteine (C) UNN
UGC UNN
none


 


UGU ANN
Cysteine (C) ANN
UGC ANN
none


 


CGU UNN
Arginine (R) UNN
CGC UNN or CGA UNN or AGA UNN or AGG 
AGG UNN




UNN



 


CGU ANN
Arginine (R) ANN
CGC ANN or CGA ANN or AGA ANN or AGG 
AGG ANN




ANN



 


AGU UNN
Serine (S) UNN
AGC UNN or UCC UNN or UCA UNN or UCG 
AGC UNN




UNN



 


AGU ANN
Serine (S) ANN
AGC ANN or UCC ANN or UCA ANN or UCG 
AGC ANN




ANN



 


GGU UNN
Glycine (G) UNN
GGA UNN or GGC UNN or GGG UNN
GGC UNN


 


GGU ANN
Glycine (G) UNN
GGA ANN or GGC ANN or GGG ANN
GGC ANN





*Human codon bias as an example of organism codon bias.


UNN: UUC, UCU, UCC, UCA, UCG,


ANN: AUC, AUG, ACU, ACC, ACA, ACG, AAU, AAC, AAA, AAG, AGU, AGC, AGA, AGG









Reference is now made to the figures, wherein
FIG. 1 is a graph showing the effect of the reduction of UU and UA dinucleotides on the expression of EGFP in a mammalian expression system in comparison to the wild type sequence,
FIG. 2 is a graph showing the effect of the reduction of UU and UA dinucleotides on the expression of EGFP in a mammalian expression system in comparison to the wild type sequence with simultaneous over-expression of RNase L, and
FIG. 3 is a graph showing the GFP fluorescence in mammalian cells transfected with PCR products harboring a modified UU/UA-reduced EGFP sequence or the wild type EGFP sequence.
FIG. 4 is a graph showing luciferase activity in Hek293 cells transfected with either a wild type or modified (i.e. UU/AG-reduced) firefly luciferase expression vector.
FIG. 5 is a graph showing luciferase activity in Huh7 cells which have been transfected with different PCR products generated from wild type or UU/UA-reduced firefly luciferase expression vector,
FIG. 6 is a graph showing expression of hepatitis B surface antigen in Hek293 cells transfected with a wild type or UU/UA-reduced hepatitis B surface antigen expression vector.
FIG. 7 is a graph showing expression of various green fluorescent proteins in Hek293 cells transfected with MODC-destabilized wildtype and MODC-destabilized UU/UA-reduced green fluorescent proteins.



The invention is now further described by reference to the following examples, which are intended to illustrate, not to limit the scope of the invention.
EXAMPLE 1
Reduction of UU and/or UA Dinucleotides in Introns
/=splicing site
Underlined=consensus functional site
Bold underlined italics=branch point







SEQ ID NO: 1: Wild type rabbit beta globin


intron 1 (RBTG1):


GGTGAGGCCGA/GTTTGGTAAGTATCCTTTTTACAGCACAACTTAAT


 


GAGACAGATAGAAACTGACCGGTGGGAGTCTGCGGCCGCAGTCTTGT


 


AGAAACAGAGTAGTCGCCTGCTTTTCTGCCAGG TTCTCT


 


CCCCTTCTCTTTTTTCCTTTTCTCAG/GTTGGTGTCG


 


SEQ ID NO: 2: UU/UA-reduced RBTG1 (without


UU/UA reduction in the CT-rich region):


GGTGAGGCCGAGTTTGGTAAGTGTCCTCTGAACAGCACAACTGAATG


 


AGAAACTGACCGGTGGGAGTCTGCGGCCGCAGTCTGTAGAAACAGA


 


GTAGTCGCCTGCTTTTCTGCCAGGTGC TTCTCTCCCCTTCTC


 


TTTTTTCCTTTTCTCAG/GTTGGTGTCG


 


SEQ ID NO: 3: UU/UA-reduced RBTG1 (with minimal


UU/UA reduction in the CT-rich region):


GGTGAGGCCGAGTTTGGTAAGTGTCCTCTGAACAGCACAACTGAATG


 


AGACAGAAGAAACTGACCGGTGGGAGTCTGCGGCCGCAGTCTGTAGA


 


AACAGAGTAGTCGCCTGTCTTCTGCCAGGTGC TCTCTCTCCC


 


CTTCTCTCTCTTCCTCTTCTCAG/GTTGGTGTCG






EXAMPLE 2
Reduction of UU and/or UA Dinucleotides in the 3′UTR
.=deletion
Underlined=mutation
Bold underlined=poly A signal







SEQ ID NO: 4: Wild type SV40 3′UTR


TGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGT


 


TACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTT


 


TTCACTGCATTCTAGTTGTGGTTTGTCCAAAC


 


SEQ ID NO: 5: UU/UA-reduced SV40 3′UTR


TGAATGCAAT.GT.GC.GTCAACT.GTCTGTCTGCAGCTCACAATGG


 


TTACAAATAAAGCAAT.GCATCACAAATCTCACAAATCAAGCATCTG


 


T..CACTGCAT.CTAGT.GTGGTCTGTCCAAAC


 


SEQ ID NO: 6: Wild type bovine growth hormone


(BGH) 3′UTR


TCTAGAGATCTGTGTGTTGGTTTTTTGTGGATCTGCTGTGCCTTCTA


 


GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACC


 


CTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAAT


 


TGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGG


 


TGGGGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT


 


GCTGCTTAAG


 


SEQ ID NO: 7: UU/UA-reduced BGH 3′UTR


TCTAGAGATCTGTGTGTTGGTCTG.TGTGGATCTGCTGTGCCT.CTA


 


GT.GCCAGCCATCTGT.GTCTGCCCCTCCCCCGTGCCT.CCT.GACC


 


CTGGAAGGTGCCACTCCCACTGTCCTGTCCTAATAAAATGAGGAAAT


 


.GCATCGCAT.GTCTGAGT.GGTGTCATCTCTATCCTGGGGGGTGGG


 


GTGGGGCAGCACAGCAAGGGGGAGGATCTGGGAAGACAAT.GCAGGC


 


ATGCTGCTTAAG






EXAMPLE 3
Reduction of UU and/or UA Dinucleotides in the Coding Region







SEQ ID NO: 8: Wild type enhanced green


fluorescent protein (EGFP)


ATGGCTAGCAAAGGAGAAGAACTCTTCACTGGAGTTGTCCCAATTCT


 


TGTTGAATTAGATGGTGATGTTAACGGCCACAAGTTCTCTGTCAGTG


 


GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTGAAGTTC


 


ATCTGCACTACTGGCAAACTGCCTGTTCCATGGCCAACACTAGTCAC


 


TACTCTGTGCTATGGTGTTCAATGCTTTTCAAGATACCCGGATCATA


 


TGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTA


 


CAGGAAAGGACCATCTTCTTCAAAGATGACGGCAACTACAAGACACG


 


TGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGT


 


TAAAAGGTATTGACTTCAAGGAAGATGGCAACATTCTGGGACACAAA


 


TTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAA


 


ACAAAAGAATGGAATCAAAGTGAACTTCAAGACCCGCCACAACATTG


 


AAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCA


 


ATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCAC


 


ACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGG


 


TCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGAT


 


GAACTGTACAAC


 


SEQ ID NO: 9: UU/UA-reduced EGFP (“SuperGFP”)


ATGGCCAGCAAGGGCGAGGAACTGTTCACCGGCGTGGTGCCCATCCT


 


GGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCG


 


GCGAGGGCGAAGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTC


 


ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGAC


 


CACCCTGTGCTACGGCGTGCAGTGCTTCAGCAGATACCCCGACCACA


 


TGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTG


 


CAGGAACGGACCATCTTCTTCAAGGACGACGGCAACTACAAGACCAG


 


GGCCGAGGTGAAGTTCGAGGGCGACACACTGGTGAACCGGATCGAGC


 


TGAAGGGCATCGACTTCAAAGAGGACGGCAACATCCTGGGCCACAAG


 


CTGGAATACAACTACAACAGCCACAACGTGTACATCATGGCCGACAA


 


GCAGAAGAACGGCATCAAGGTCAACTTCAAGACCCGGCACAACATCG


 


AGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCC


 


ATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC


 


CCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGGGACCACATGG


 


TGCTGCTGGAATTCGTGACAGCCGCCGGAATCACCCACGGCATGGAC


 


GAGCTGTACAAC


SEQ ID NO: 10


WILD TYPE GFP SEQUENCE FROM MONTASTREA


CAVERNOSA


ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGGATGGAGGG


 


CGCCGTGAACGGCCACAAATTCGTGATCGAGGGCGACGGGAAAGGCA


 


AGCCCTTTGAGGGTAAGCAGACTATGGACCTGACCGTGATCGAGGGC


 


GCCCCCCTGCCCTTCGCTTATGACATTCTCACCACCGTGTTCGACTA


 


CGGTAACCGTGTCTTCGCCAAGTACCCCAAGGACATCCCTGACTACT


 


TCAAGCAGACCTTCCCCGAGGGCTACTCGTGGGAGCGAAGCATGACA


 


TACGAGGACCAGGGAATCTGTATCGCTACAAACGACATCACCATGAT


 


GAAGGGTGTGGACGACTGCTTCGTGTACAAAATCCGCTTCGACGGGG


 


TCAACTTCCCTGCTAATGGCCCGGTGATGCAGCGCAAGACCCTAAAG


 


TGGGAGCCCAGTACCGAGAAGATGTACGTGCGGGACGGCGTACTGAA


 


GGGCGATGTTAATATGGCACTGCTCTTGGAGGGAGGCGGCCACTACC


 


GCTGCGACTTCAAGACCACCTACAAAGCCAAGAAGGTGGTGCAGCTT


 


CCCGACTACCACTTCGTGGACCACCGCATCGAGATCGTGAGCCACGA


 


CAAGGACTACAACAAAGTCAAGCTGTACGAGCACGCCGAAGCCCACA


 


GCGGACTACCCCGCCAGGCCGGCTAA


 


SEQ ID NO: 11


MONSTER-OM: MODIFIED TYPE OF SEQ ID NO: 10 BY


REDUCING THE NUMBER OF UU/UA DINUCLEOTIDES


ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGGATGGAGGG


 


CGCCGTGAACGGCCACAAATTCGTGATCGAGGGCGACGGGAAAGGCA


 


AGCCCTTCGAGGGCAAGCAGACGATGGACCTGACCGTGATCGAGGGC


 


GCCCCCCTGCCCTTCGCCTACGACATCCTGACCACCGTGTTCGACTA


 


CGGCAACCGTGTCTTCGCCAAGTACCCCAAGGACATCCCTGACTACT


 


TCAAGCAGACCTTCCCCGAGGGCTACTCGTGGGAGCGAAGCATGACA


 


TACGAGGACCAGGGAATCTGCATCGCGACAAACGACATCACCATGAT


 


GAAGGGTGTGGACGACTGCTTCGTGTACAAAATCCGCTTCGACGGGG


 


TCAACTTCCCTGCCAATGGCCCGGTGATGCAGCGCAAGACCCTGAAG


 


TGGGAGCCCAGCACCGAGAAGATGTACGTGCGGGACGGCGTCCTGAA


 


GGGCGATGTGAACATGGCACTGCTCCTGGAGGGAGGCGGCCACTACC


 


GCTGCGACTTCAAGACCACCTACAAAGCCAAGAAGGTGGTGCAGCTG


 


CCCGACTACCACTTCGTGGACCACCGCATCGAGATCGTGAGCCACGA


 


CAAGGACTACAACAAAGTCAAGCTGTACGAGCACGCCGAAGCCCACA


 


GCGGACTGCCCCGCCAGGCCGGCTGAAGTCTCACGGCTTCCCACCCG


 


AGGTCGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAG


 


GAGTCTGGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGAT


 


CAATGTG TGA


 


SEQ ID NO: 12


CLAVULARIA SPECIES-WILD TYPE SEQUENCE


ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGA


 


CATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCT


 


TCGTGATCGAGGGC GAGGGCGAGGGCAAGCCCTACGACGGCACCAA


 


CACATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTA


 


CGACATTCTGACCACCGCGTTCAGTTACGGCAACAGGGCCTTCACCA


 


AGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAG


 


GGCTACTCTTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGT


 


GAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACG


 


AGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATG


 


CAGAAGGAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGT


 


GCGCGACGGCGTGCTGAAGGGCGACGTCAAGATGAAGCTGCTGCTGG


 


AGGGCGGCGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCC


 


AAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTGGACCACCGCAT


 


CGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACG


 


AGATCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTAC


 


AAGTAA


 


SEQ ID NO.: 13


CLAVULARIA SPECIES-OM: MODIFIED


(UU/UA-reduced)


ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTGATCAAGCCCGA


 


CATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCT


 


TCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAAC


 


ACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTA


 


CGACATCCTGACCACCGCGTTCAGCTACGGCAACAGGGCCTTCACCA


 


AGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAG


 


GGCTACAGCTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGT


 


GAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACG


 


AGATCCACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATG


 


CAGAAGGAGACCACCGGCTGGGACGCCTCCACCGAGAGGATGTACGT


 


GCGCGACGGCGTGCTGAAGGGCGAGCGTCAAGATGAAGCTGCTGCTG


 


GAGGGCGGCGGCCACCACCGCGTGGACTTCAAGACCATCTACAGGGC


 


CAAGAAGGCGGTGAAGCTGCCCGACTATCACTTCGTGGACCACCGCA


 


TCGAGATCCTGAACCACGACAAGGACTACAACAAGGTACCGTGTACG


 


AGATCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCTGTAC


 


AAGCTGA


 


SEQ ID NO: 14


FIREFLY LUCIFERASE +: WILD TYPE SEQUENCE:


ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCC


 


GCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGA


 


GATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATC


 


GAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTT


 


GGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCG


 


TCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGC


 


GCGTTATTTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATA


 


ATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTG


 


GTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAA


 


AAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGG


 


ATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTA


 


CCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAG


 


GGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTC


 


TGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTC


 


TCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATAC


 


TGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTA


 


CTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTAT


 


AGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGAT


 


TCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAA


 


GCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCT


 


TCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAA


 


GAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGA


 


CTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGC


 


GCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCT


 


GGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTG


 


TGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCG


 


ACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACAT


 


AGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGA


 


AGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTG


 


GAATCC


 


SEQ ID NO: 15


LUC + DU (Superluciferase): (UU/UA-reduced)


ATGGAAGACGCCAAAAACATCAAGAAAGGCCCGGCGCCATTCTACCC


 


GCTGGAAGATGGAACCGCTGGAGAGCAACTGCACAAGGCCATGAAGA


 


GATACGCCCTGGTGCCTGGAACAATCGCGTTCACAGATGCACACATC


 


GAGGTGGACATCACCTACGCTGAGTACTTCGAAATGTCCGTCCGGCT


 


GGCAGAAGCCATGAAACGATACGGGCTGAACACAAATCACAGATCGT


 


CGTGTGCAGTGAAAACTCTCTGCAATTCTTCATGCCGGTGCTGGGCG


 


CGCTGTTCATCGGAGTGGCAGTCGCGCCCGCGAACGACATCTACAAT


 


GAACGTGAACTCCTCAACAGCATGGGCATCTCGCAGCCCACCGTGGT


 


GTTCGTGTCCAAAAAGGGGCTGCAAAAAATCCTGAACGTGCAAAAAA


 


AGCTCCCAATCATCCAAAAAATCATCATCATGGACAGCAAAACGGAC


 


TACCAGGGATTCCAGTCGATGTACACGTTCGTCACATCTCATCTGCC


 


TCCCGGCTTCAATGAATACGACTTCGTGCCAGAGTCCTTCGACAGGG


 


ACAAGACAATCGCACTGATCATGAACTCCTCTGGAAGCACTGGTCTG


 


CCCAAAGGTGTCGCTCTGCCTCACAGAACTGCCTGCGTGAGATTCTC


 


GCATGCCAGAGATCCCATCTTCGGCAATCAAATCATCCCGGACACTG


 


CGATCCTGAGTGTGGTCCCATTCCATCACGGCTTCGGAATGTTCACG


 


ACACTCGGATACCTGATCTGTGGATTCCGAGTCGTCCTGATGTACAG


 


ATTCGAAGAAGAGCTGTTCCTGAGGAGCCTCCAGGACTACAAGATCC


 


AAAGTGCGCTGCTGGTGCCAACCCTGTTCTCCTTCTTCGCCAAAAGC


 


ACTCTGATCGACAAATACGATCTCAGCAATCTGCACGAAATCGCCTC


 


TGGTGGCGCTCCCCTCTCCAAGGAAGTCGGGGAAGCGGTCGCCAAGA


 


GGTTCCATCTGCCAGGGATCAGGCAAGGATACGGGCTCACTGAGACG


 


ACATCAGCCATCCTGATCACACCCGAGGGGGATGACAAACCGGGCGC


 


GGTCGGGAAAGTGGTCCCATTCTTCGAAGCGAAGGTTGTGGATCTGG


 


ACACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTG


 


AGAGGTCCCATGATCATGTCCGGCTACGTGAACAATCCGGAAGCGAC


 


CAACGCCCTGATCGACAAGGATGGATGGCTCCACTCTGGAGACATCG


 


CGTACTGGGACGAAGACGAACACTTCTTCATCGTGGACCGCCTGAAG


 


TCTCTGATCAAGTACAAAGGCTACCAGGTGGCTCCCGCTGAACTCGA


 


ATCCATCCTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAG


 


GTCTGCCCGACGATGACGCCGGTGAACTGCCCGCCGCCGTCGTGGTT


 


CTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGACTACGT


 


CGCCAGTCAAGTAACAACCGCGAAAAAGCTGCGCGGAGGAGTTGTGT


 


TCGTGGACGAAGTGCCGAAAGGTCTGACCGGAAAACTCGACGCAAGA


 


AAAATCAGAGAGATCCTCATCAAGGCCAAGAAGGGCGGAAAGATCGC


 


CGTG


 


SEQ ID NO: 16


Firefly LUC2 luciferase wildtype:


ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCC


 


ACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGC


 


GCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATC


 


GAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCT


 


GGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCG


 


TGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGT


 


GCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAA


 


CGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCG


 


TATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAG


 


AAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGA


 


CTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGC


 


CACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGG


 


GACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATT


 


GCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCA


 


GTCATGCCCGCGACCCCATCTTTCGGCAACCAGATCATCCCCGACAC


 


CGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCA


 


CCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTAC


 


CGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACCTATAAGA


 


TTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAG


 


AGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGC


 


CAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCA


 


AACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAA


 


ACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGG


 


CGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACT


 


TGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGC


 


GTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGC


 


TACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACA


 


TCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTG


 


AAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACT


 


GGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCG


 


CCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTC


 


GTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTA


 


TGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTG


 


TGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCC


 


CGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGAT


 


CGCCGTGTAATAA


 


SEQ ID NO: 17


LUC2OM: LUC2 modified SuperLuciferase2


(UU/UA-reduced)


ATGGAAGATGCCAAAAACATCAAGAAGGGCCCAGCGCCATTCTACCC


 


ACTCGAAGACGGGACCGCAGGCGAGCAGCTGCACAAAGCCATGAAGC


 


GCTACGCCCTGGTGCCCGGCACCATCGCCTTCACCGACGCACACATC


 


GAGGTGGACATCACCTACGCCGAGTACTTCGAGATGAGCGTGCGGCT


 


GGCAGAAGCCATGAAGCGCTACGGGCTGAACACAAACCATCGGATCG


 


TGGTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCCGTGCTGGGT


 


GCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCCAACGACATCTACAA


 


CGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCG


 


TGTTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAG


 


AAGCTGCCGATCATCCAAAAGATCATCATCATGGACAGCAAGACCGA


 


CTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACCTCCCACCTGC


 


CACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGG


 


GACAAAACCATCGCCCTGATCATGAACAGCAGTGGCAGCACCGGACT


 


GCCCAAGGGCGTGGCACTGCCGCACCGCACCGCCTGTGTCCGATTCA


 


GTCATGCACGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACC


 


GCCATCCTCAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCAC


 


CACGCTGGGCTACTGGATCTGCGGCTTCCGGGTCGTGCTCATGTACC


 


GCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAGATC


 


CAATCTGCCCTGCTGGTGCCCACACTGTTCAGCTTCTTCGCCAAGAG


 


CACTCTCATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCCA


 


GCGGCGGAGCGCCGCTCAGCAAGGAGGTGGGTGAGGCCGTGGCCAAA


 


CGCTTCCACCTGCCAGGCATCCGCCAGGGCTACGGCCTGACAGAAAC


 


AACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCG


 


CAGTGGGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTG


 


GACACCGGCAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGT


 


CCGTGGCCCCATGATCATGAGCGGCTACGTGAACAACCCCGAGGCCA


 


CAAACGCTCTCATCGACAAGGACGGCTGGCTGCACACGCGGCGACAT


 


CGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGA


 


AGAGCCTGATCAAATACAAGGGCTACCAGGTGGCCCCAGCCGAACTG


 


GAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGAGTCGC


 


CGGACTGCCAGACGACGATGCCGGCGAGCTGCCCGCAGCAGTCGTCG


 


TGCTGGAACACGGCAAAACCATGACCGAGAAGGAGATCGTGGACTAC


 


GTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGTGGTGTGGT


 


GTTCGTGGACGAGGTGCCCAAAGGACTGACCGGCAAGCTGGACGCCC


 


GCAAGATCCGCGAGATCCTCATCAAGGCCAAGAAGGGCGGCAAGATC


 


GCCGTGTGA


 


SEQ ID NO: 18


Puntellina Plumate (GFP) wild type:


ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCG


 


CATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCG


 


GAGAGGGCACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAGAGC


 


ACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT


 


GGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGA


 


ACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGC


 


ATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTA


 


CCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGATGGGCA


 


CCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGC


 


AGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGATAACGATCT


 


GGATGGCAGCTTCACCCGCACCTTCAGCCTGCGCGACGGCGGCTACT


 


ACAGCTCCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCAC


 


CCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGT


 


GGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGC


 


ACGCCTTCAAGACCCCGGATGCAGATGCCGGTGAAGAAA


 


SEQ ID NO: 19


Puntellina Plumate (GFP): modified sequence


(UU/UA-reduced)


ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCG


 


CATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCG


 


GAGAGGGCACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAGAGC


 


ACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT


 


GGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGA


 


ACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGC


 


ATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCATCTA


 


CCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGATGGGCA


 


CCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGC


 


AGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGACAACGACCT


 


GGATGGCAGCTTCACCCGCACCTTCAGCCTGCGCGACGGCGGCTACT


 


ACAGCTCCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCAC


 


CCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGT


 


GGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGAGATACCAG


 


CACGCCTTCAAGACCCCGGATGCAGATGCCGGTGAAGAACTGA


 


SEQ ID NO: 20


Red Fluorescent protein from Discosoma wild


type sequence


atgagcgagctgatcaaggagaacatgcacatgaagctgtacatgga


 


gggcaccgtgaacaaccaccacttcaagtgcacatccgagggcgaag


 


gcaagccctacgagggcacccagaccatgaagatcaaggtggtcgag


 


ggcggccctctccccttcgccttcgacatcctggctaccagcttcat


 


gtacggcagcaaagccttcatcaaccacacccagggcatccccgact


 


tctttaagcagtccttccctgagggcttcacatgggagagaatcacc


 


acatacgaagacgggggcgtgctgaccgctacccaggacaccagctt


 


ccagaacggctgcatcatctacaacgtcaagatcaacggggtgaact


 


tcccatccaacggccctgtgatgcagaagaaaacacgcggctgggag


 


gccaacaccgagatgctgtaccccgctgacggcggcctgagaggcca


 


cagccagatggccctgaagctcgtgggcgggggctacctgcactgct


 


ccttcaagaccacatacagatccaagaaacccgctaagaacctcaag


 


atgcccggcttccacttcgtggaccacagactggaaagaatcaagga


 


ggccgacaaagagacctacgtcgagcagcacgagatggctgtggcca


 


agtactgcgacctccctagcaaactggggcacagagatga


 


SEQ ID NO: 21


Red Fluorescent protein modified sequence


ATGAGCGAGCTGATCAAGGAGAACATGCACATGAAGCTGTACATGGA


 


GGGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAG


 


GCAAGCCCTACGAGGGCACCCAGACCATGAAGATCAAGGTGGTCGAG


 


GGCGGCCCACTCCCCTTCGCCTTCGACATCCTGGCCACCAGCTTCAT


 


GTACGGCAGCAAAGCCTTCATCAACCACACCCAGGGCATCCCCGACT


 


TCTTCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAATCACC


 


ACATACGAAGACGGGGGCGTGCTGACCGCACCCAGGACACCAGCTTC


 


CAGAACGGCTGCATCATCTACAACGTCAAGATCAACGGGGTGAACTT


 


CCCATCCAACGGCCCTGTGATGCAGAAGAAAACACGCGGCTGGGAGG


 


CCAACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGAGAGGCCAC


 


AGCCAGATGGCCCTGAAGCTCGTGGGCGGGGGCTACCTGCACTGCTC


 


CTTCAAGACCACATACAGATCCAAGAAACCCGCCAAGAACCTCAAGA


 


TGCCCGGCTTCCACTTCGTGGACCACAGACTGGAAAGAATCAAGGAG


 


GCCGACAAAGAGACCTACGTCGAGCAGCACGAGATGGCTGTGGCCAA


 


GTACTGCGACCTCCCAAGCAAACTGGGGCACAGAC


 


SEQ ID NO: 22


Hepatitis B surface antigen wild type, adr


hepatitis B virus strain:


ATGGAGAACACAACATCAGGATTCCTAGGACCCCTGCTCGTGTTACA


 


GGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCACAGAGTC


 


TAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAGCACCCACG


 


TGTCCTGGCCCAAATTCGCAGTCCCCAACCTCCAATCACTCACCAAC


 


CTCTTGTCCTCCAATTTGTCCTGGCTATTCGCTGGATGTGTCTGCGG


 


CGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTT


 


GTTGGTTCTTCTGGACTACCAAGGTATGTTGCCCGTTTGTCCTCTAC


 


TTCCAGGAACATCAACTACCAGCACGGGACCATGCAAGACCTGCACG


 


ATTCCTGCTCAAGGAACCTCTATGTTTCCCTCCTGTTGCTGTACAAA


 


ACCTTCGGACGGAAACTGCACTTGTATTCCCATCCCATCATCCTGGG


 


CTTTCGCAAGATTCCTATGGGAGTGGGCCTCAGTCCGTTTCTCCTGG


 


CTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCC


 


CACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAA


 


GTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTC


 


TTTTGTCTTTGGGTATACATTTGA


 


SEQ ID NO: 23


HBSAGOM: Hepatitis B surface antigen modified


sequence:


ATGGAGAACACCACCAGCGGCTTCCTGGGCCCTCTGCTGGTGCTGCA


 


GGCCGGCTTCTTCCTGCTGACCCGCATCCTGACCATCCCCCAGAGCC


 


TGGACAGCTGGTGGACCAGCCTGAACTTCCTGGGCGGAGCCCCAACC


 


TGTCCCGGCCCCAACAGCCAGAGCCCCACCAGCAACCACAGCCCAAC


 


CAGCTGCCCACCCATCTGTCCCGGCTACCGGTGGATGTGCCTGCGGC


 


GGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTC


 


CTGGTGCTCCTGGACTACCAGGGCATGCTGCCCGTGTGTCCTCTGCT


 


GCCTGGCACCAGCACCACCTCCACCGGCCCCTGCAAGACCTGCACAA


 


TCCCCGCCCAGGGAACCAGCATGTTCCCAAGCTGCTGCTGCACCAAG


 


CCCAGCGACGGCAACTGCACCTGCATCCCCATCCCAAGCAGCTGGGC


 


CTTCGCCAGATTCCTGTGGGAGTGGGCCTCCGTGAGATTCAGCTGGC


 


TGTCACTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCA


 


ACAGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGGGGACCCAG


 


CCTGTACAACATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTCT


 


TCTGCCTGTGGGTGTACATCTGA


 


SEQ ID NO: 24


HBSAGM: HBSAGOM: Hepatitis B surface antigen


modified sequence 2


ATGGAGAACACAACATCAGGATTCCTCGGACCCCTGCTCGTGCTGCA


 


GGCGGGGTTCTTCCTGCTCACAAGAATCCTCACAATCCCACAGAGTC


 


TGGACTCGTGGTGGACGTCTCTCAACTTCCTCGGGGGAGCACCCACG


 


TGTCCTGGCCCAAACTCGCAGTCCCCAACCTCCAATCACTCACCAAC


 


CTCGTGTCCTCCAATCTGTCCTGGCTACCGCTGGATGTGTCTGCGGC


 


GCTTCATCATCTTCCTCTTCATCCTGCTGCTGTGCCTCATCTTCCTG


 


CTCGTCCTCCTGGACTACCAAGGGATGCTGCCCGTCTGTCCTCTGCT


 


GCCAGGAACATCAACCACCAGCACGGGACCATGCAAGACCTGCACGA


 


TCCCTGCTCAAGGAACCAGCATGTTCCCCTCCTGCTGCTGCACAAAA


 


CCATCGGACGGAAACTGCACCTGCATCCCCATCCCATCATCCTGGGC


 


CTTCGCAAGATTCCTCTGGGAGTGGGCCTCAGTCCGGTTCTCCTGGC


 


TCAGTCTCCTGGTGCCATTCGTGCAGTGGTTCGTCGGGCTGTCCCCC


 


ACTGTGTGGCTGTCAGTGATCTGGATGATGTGGTACTGGGGGCCAAG


 


TCTGTACAACATCCTCAGTCCCTTCCTGCCTCTGCTGCCAATCTTCT


 


TCTGTCTGTGGGTGTACATCTGA


 


This sequence includes both an UU/UA reduction


as well as a humanization.


SEQ ID NO: 25


IFN-ALPHA, HUMAN


ATGGCCTTGACCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTG


 


CAAGTCAAGCTGCTCTGTGGGCTGTGATCTGCCTCAAACCCACAGCC


 


TGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATC


 


TCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCA


 


GGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCC


 


TCCATGAGATGATCCAGCAGATCTTCAATCTCTTCAGCACAAAGGAC


 


TCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGA


 


ACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGG


 


TGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCT


 


GTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAA


 


ATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGAT


 


CTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA


 


TGA


 


SEQ ID NO: 26


IFN-ALPHAOM: modified sequence


ATGGCCCTGACCTTCGCCCTGCTGGTGGCTCTGCTGGTGCTGAGCTG


 


CAAGAGCAGCTGCAGCGTGGGCTGCGATCTGCCTCAGACCCACAGCC


 


TGGGCAGCAGACGGACACTGATGCTGCTGGCCCAGATGCGGCGGATC


 


AGCCTGTTCAGCTGCCTGAAGGACCGGCACGACTTCGGCTTCCCCCA


 


GGAAGAGTTCGGCAACCAGTTCCAGAAGGCCGAGACAATCCCCGTGC


 


TGCACGAGATGATCCAGCAGATCTTCAACCTGTTCAGCACCAAGGAC


 


AGCAGCGCCGCCTGGGACGAGACACTGCTGGACAAGTTCTACACCGA


 


GCTGTACCAGCAGCTGAACGACCTGGAAGCCTGCGTGATCCAGGGCG


 


TGGGCGTGACCGAGACACCCCTGATGAAGGAAGACAGCATCCTGGCC


 


GTGCGGAAGTACTTCCAGCGGATCACCCTGTACCTGAAAGAGAAGAA


 


GTACAGCCCCTGCGCCTGGGAAGTGGTCCGGGCCGAGATCATGCGGA


 


GCTTCAGCCTGAGCACCAACCTGCAGGAAAGCCTGCGGAGCAAAGAG


 


ATGA


 


SEQ ID NO: 27


CSF3M Colony stimulating factor wild type


sequence, human:


ATGGCTGGACCCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGC


 


AGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACC


 


CCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTG


 


CTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGG


 


AGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTG


 


CTGCTCGGACACTCTCTCGGGCATCCCCTGGGCTCCCCTGAGCAGCT


 


GCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCAT


 


AGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGAT


 


CTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCG


 


CCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATG


 


GCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTC


 


TGCTTTCCAGCGCCGGGCAGGAGGGGTCCTAGTTGCCTCCCATCTGC


 


AGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAG


 


CCC


 


SEQ ID NO: 28


CSF3M Colony stimulating factor modified


sequence,:


ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCA


 


GCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCC


 


CCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGC


 


CTGGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCCAGCGCTCCAGG


 


AGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTG


 


CTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTG


 


CCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCCTGAGCCAACTCCACA


 


GCGGCCTCTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATC


 


TCCCCCGAGCTGGGTCCCACCCTGGACACACTGCAGCTGGACGTCGC


 


CGACTTCGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGG


 


CCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCT


 


GCCTTCCAGCGCCGGGCAGGAGGGGTCCTGGTGGCCTCCCATCTGCA


 


GAGCTTCCTGGAGGTGTCGTACCGCGTGCTCCGCCACCTCGCCCAGC


 


CC


 


SEQ ID NO: 29


MODC wild type sequence (mouse ornithine


decarboxylase)


CAGAGCCATGGCTTCCCGCCGGAGGTGGAGGAGCAGGATGATGGCAC


 


GCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTG


 


CAGCCTGTGCTTCTGCTAGGATCAATGTG


 


 


SEQ ID NO: 30


MODC modified sequence:


AGTCTCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATGATGGCACA


 


CTGCCCATGAGCTGTGCTCAGGAGTCTGGCATGGACAGACACCCCGC


 


TGCCTGTGCCAGTGCCAGGATCAATGTG TGA






Destabilized and sequence modified reporters:

EXAMPLES OF destabilized and modified REPORTER SEQUENCES:








SEO ID NO: 31


MONTASTRAEA CAVERNOSA


ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGGATGGAGGG


 


CGCCGTGAACGGCCACAAATTCGTGATCGAGGGCGACGGGAAAGGCA


 


AGCCCTTCGAGGGCAAGCAGACCGATGGACCTGACCGTGATCGAGGG


 


CGCCCCCCTGCCCTTCGCCTACGACATCCTGACCACCGTGTTCGACT


 


ACGGCAACCGTGTCTTCGCCAAGTACCCCAAGGACATCCCTGACTAC


 


TTCAAGCAGACCTTCCCCGAGGGCTACTCGTGGGAGCGAAGCATGAC


 


ATACGAGGACCAGGGAATCTGCATCGCGACAAACGACATCACCATGA


 


TGAAGGGTGTGGACGACTGCTTCGTGTACAAAATCCGCTTCGACGGG


 


GTCAACTTCCCTGCCAATGGCCCGGTGATGCAGCGCAAGACCCTGAA


 


GTGGGAGCCCAGCACCGAGAAGATGTACGTGCGGGACGGCGTCCTGA


 


AGGGCGATGTGAACATGGCACTGCTCCTGGAGGGAGGCGGCCACTAC


 


CGCTGCGACTTCAAGACCACCTACAAAGCCAAGAAGGTGGTGCAGCT


 


GCCCGACTACCACTTCGTGGACCACCGCATCGAGATCGTGAGCCACG


 


ACAAGGACTACAACAAAGTCAAGCTGTACGAGCACGCCGAAGCCCAC


 


AGCGGACTGCCCCGCCAGGCCGGCAGTCTCACGGCTTCCCACCCGAG


 


GTCGAGGAGCAGGATGATGGCACACTGCCCATGAGCTGTGCTCAGGA


 


GTCTGGCATGGACAGACACCCCGCTGCCTGTGCCAGTGCCAGGATCA


 


ATGTGTGA


 


Bold in MODC seqeuence.


 


SEQ ID NO: 32


Clavulariidae Clavularia-OM: MODIFIED


ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTGATCAAGCCCGA


 


CATGAAGATCAAGCTGAAGATGGAGGGCAACGTGAATGGCCACGCCT


 


TCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAAC


 


ACCATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTA


 


CGACATCCTGACCACCGCGTTCAGCTACGGCAACAGGGCCTTCACCA


 


AGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAG


 


GGCTACAGCTGGGAGCGCACCATGACCTTCGAGGACAAGGGCATCGT


 


GAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATCTACG


 


AGATCCACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATG


 


CAGAAGGAGACCACCGGCTGGGACGCCTCCACCGAGAAGGGATGTAC


 


GTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGATTGAAGCTGCTGC


 


TGGAGGGCGGCGGCCACCACCGCGTGGACTTCAAGACCATCTACAGG


 


GCCAAGAAGGCCGGTGAAGCTGCCCGACTATCACTTCGTGGACCACC


 


GCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTG


 


TACGAGATCGCCGTGGCCCGCAACTCCACCGACGGCATGGACGAGCT


 


GTACAAGCAGTCTCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATG


 


ATGGCACACTGCCCCATGAGCTGTGCTCAGGAGTCTGGCATGGACAG


 


ACACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA


 


SEQ ID NO: 33


Firefly LUC + DU (Superluciferase):


ATGGAAGACGCCAAAAACATCAAGAAAGGCCCGGCGCCATTCTACCC


 


GCTGGAAGATGGAACCGCTGGAGAGCAACTGCACAAGGCCATGAAGA


 


GATACGCCCTGGTGCCTGGAACAATCGCGTTCACAGATGCACACATC


 


GAGGTGGACATCACCTACGCTGAGTACTTCGAAATGTCCGTCCGGCT


 


GGCAGAAGCCATGAAACGATACGGGCTGAACACAAATCACAGAATCG


 


TCGTGTGCAGTGAAAACTCTCTGCAATTCTTCATGCCGGTGCTGGGC


 


GCGCTGTTCATCGGAGTGGCAGTCGCGCCCGCGAACGACATCTACAA


 


TGAACGTGAACTCCTCAACAGCATGGGCATCTCGCAGCCCACCGTGG


 


TGTTCGTGTCCAAAAAGGGGCTGCAAAAAATCCTGAACGTGCAAAAA


 


AAGCTCCCAATCATCCAAAAAATCATCATCATGGACAGCAAAACGGA


 


CTACCAGGGATTCCAGTCGATGTACACGTTCGTCACATCTCATCTGC


 


CTCCCGGCTTCAATGAATAACGACTTCGTGCCAGAGTCCTTCGACAG


 


GGACAAGACAATCGCACTGATCATGAACTCCTCTGGAAGCACTGGTC


 


TGCCCAAAGGTGTCGCTCTGCCTCACAGAACTGCCTGCGTGAGATTC


 


TCGCATGCCAGAGATCCCATCTTCGGCAATCAAATCATCCCGGACAC


 


TGCGATCCTGAGTGTGGTCCCATTCCATCACGGCTTCGGAATGTTCA


 


CGACACTCGGATACCTGATCTGTGGATTCCGAGTCGTCCTGATGTAC


 


AGATTCGAAGAAGAGCTGTTCCTGAGGAGCCTCCAGGACTACAAGAT


 


CCAAAGTGCGCTGCTGGTGCCAACCCTGTTCTCCTTCTTCGCCAAAA


 


GCACTCTGATCGACAAATACGATCTCAGCAATCTGCACGAAATCGCC


 


TCTGGTGGCGCTCCCCTCTCCAAGGAAGTCGGGGAAGCGGTCGCCAA


 


GAGGTTCCATCTGCCAGGGATCAGGCAAGGATACGGGCTCACTGAGA


 


CGACATCAGCCATCCTGATCACACCCGAGGGGGATGACAAACCGGGC


 


GCGGTCGGGAAAGTGGTCCCCATTCTTCGAAGCGAAGGTTGTGGATC


 


TGGACACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGT


 


GTGAGAGGTCCCATGATCATGTCCGGCTACGTGAACAATCCGGAAGC


 


GACCAACGCCCTGATCGACAAGGATGGATGGCTCCACTCTGGAGACA


 


TCGCGTACTGGGACGAAGACGAACACTTCTTCATCGTGGACCGCCTG


 


AAGTCTCTGATCAAGTACAAAGGCTACCAGGTGGCTCCCGCTGAACT


 


CGAATCCATCCTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCG


 


CAGGTCTGCCCGACGATGACGCCGGTGAACTGCCCGCCGCCGTCGTG


 


GTTCTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGACTA


 


CGTCGCCAGTCAAGTAACAACCGCGAAAAAGCTGCGCGGAGGAGTTG


 


TGTTCGTGGACGAAGTGCCGAAAGGTCTGACCGGAAAACTCGACGCA


 


AGAAAAATCAGAGAGATCCTCATCAAGGCCAAGAAGGGCGGAAAGAT


 


CGCCGTGAGTCTCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATGA


 


TGGCACACTGCCCATGAGCTGTGCTCAGGAGTCTGGCATGGACAGAC


 


ACCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA


 


SEQ ID NO: 34


Firefly LUC2OM: LUC2 modified SuperLuciferase2


ATGGAAGATGCCAAAAACATCAAGAAGGGCCCAGCGCCATTCTACCC


 


ACTCGAAGACGGGACCGCAGGCGAGCAGCTGCACAAAGCCATGAAGC


 


GCTACGCCCTGGTGCCCGGCACCATCGCCTTCACCGACGCACACATC


 


GAGGTGGACATCACCTACGCCGAGTACTTCGAGATGAGCGTGCGGCT


 


GGCAGAAGCCATGAAGCGCTACGGGCTGAACACAAACCATCGGATCG


 


TGGTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCCGTGCTGGGT


 


GCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCCAACGACATCTACAA


 


CGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCG


 


TGTTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAG


 


AAGCTGCCGATCATCCAAAAGATCATCATCATGGACAGCAAGACCGA


 


CTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACCTCCCACCTGC


 


CACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGG


 


GACAAAACCATCGCCCTGATCATGAACAGCAGTGGCAGCACCGGACT


 


GCCCAAGGGCGTGGCACTGCCGCACCGCACCGCCTGTGTCCGATTCA


 


GTCATGCACGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACC


 


GCCATCCTCAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCAC


 


CACGCTGGGCTACTGGATCTGCGGCTTCCGGGTCGTGCTCATGTACC


 


GCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAGATC


 


CAATCTGCCCTGCTGGTGCCCACACTGTTCAGCTTCTTCGCCAAGAG


 


CACTCTCATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCCA


 


GCGGCGGAGCGCCGCTCAGCAAGGAGTGGGTGAGGCCGTGGCCAAAC


 


GCTTCCACCTGCCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACA


 


ACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGC


 


AGTGGGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTGG


 


ACACCGGCAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTC


 


CGTGGCCCCATGATCATGAGCGGCTACGTGAACAACCCCGAGGCCAC


 


AAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCG


 


CCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAG


 


AGCCTGATCAAATACAAGGGCTACCAGGTGGCCCCAGCCGAACTGGA


 


GAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGAGTCGCCG


 


GACTGCCAGACGACGATGCCGGCGAGCTGCCCCGCAGCAGTCGTCGT


 


GCTGGAAACACGGCAAAACCATGACCGAGAAGGAGATCGTGGACTAC


 


GTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGTGGTGTGGT


 


GTTCGTGGACGAGGTGCCCAAAGGACTGACCGGCAAGCTGGACGCCC


 


GCAAGATCCGCGAGATCCTCATCAAGGCCAAGAAGGGCGGCAAGATC


 


GCCGTGAGTCTCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATGAT


 


GGCACACTGCCCATGAGCTGTGCTCAGGAGTCTGGCATGGACAGACA


 


CCCCGCTGCCTGTGCCAGTGCCAGGATCAATGTG TGA


 


SEQ ID NO: 35


Puntellina Plumate GFP: modified sequences


ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCGAGTGCCG


 


CATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCG


 


GAGAGGGCACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAGAGA


 


CACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGA


 


TGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAG


 


AACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCG


 


CATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGTAGCTTCAGC


 


TACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGATGGG


 


CACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCC


 


GCAGCAACGCCACCGTGGAGCACCTGCACCCCCATGGGCGACAACGA


 


CCTGGATGGCAGCTTCACCCGCACCTTCAGCCTGCGCGACGGCGGCT


 


ACTACAGCTCCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATC


 


CACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCG


 


CGTGGAGGAGGATCACAGCAACACCGAGCTGGGCATCGTGGAGTACC


 


AGCACGCCTTCAAGACDCCCGGATGCAGATGCCGGTGAAGAACAGTC


 


TCACGGCTTCCCACCCGAGGTCGAGGAGCAGGATGATGGCACACTGC


 


CCATGAGCTGTGCTCAGGAGTCTGGCATGGACAGACACCCCGCTGCC


 


TGTGCCAGTGCCAGGATCAATGTG TGA


 


SEQ ID NO: 36


Red Fluorescent protein modified sequence


ATGAGCGAGCTGATCAAGGAGAACATGCACATGAAGCTGTAATGGAG


 


GGCACCGTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGG


 


CAAGCCCTACGAGGGCACCCAGACCATGAAGATCAAGGTGGTCGAGG


 


GCGGCCCACTCCCCTTCGCCTTCGACATCCTGGCCACCAGCTTCATG


 


TACGGCAGCAAAGCCTTCATCAACCACACCCAGGGCATCCCCGACTT


 


CTTCAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAATCACCA


 


CATACGAAGACGGGGGCGTGCTGACCGCCACCCAGGACACCAGCTTC


 


CAGAACGGCTGCATCATCTACAACGTCAAGATCAACGGGGTGAACTT


 


CCCATCCAACGGCCCTGTGATGCAGAAGAAAACACGCGGCTGGGAGG


 


CCAACACCGAGATGCTGTACCCCGCTGACGGCGGCCTGAGAGGCCAC


 


AGCCAGATGGCCCTGAAGCTCGTGGGCGGGGGCTACCTGCACTGCTC


 


CTTCAAGACCACATACAGATCCAAGAAACCCGCCAAGAACCTCAAGA


 


TGCCCGGCTTCCACTTCGTGGACCACAGACTGGAAAGAATCAAGGAG


 


GCCGACAAAAGAGACCTACGTCGAGCAGCACGAGATGGCTGTGGCCA


 


AGTACTGCGACCTCCCAAGCAAACTGGGGCACAGACAGTCTCACGGC


 


TTCCCACCCGAGGTCGAGGAGCAGGATGATGGCACACTGCCCATGAG


 


CTGTGCTCAGGAGTCTGGCATGGACAGACACCCCGCTGCCTGTGCCA


 


GTGCCAGGATCAATGT TGA






EXAMPLE 4
In Vivo Analysis of UU/UA-Reduced EGFP
(1) The modified EGFP sequence was custom synthesized by a gene synthesis company and supplied contained in a pUC19 vector with flanking SalI and BamHI sites. 10 μg of the vector were digested with 10 units of Sail in a buffer containing 0.1 μg/ml BSA for 1 hr at 37° C., followed by digestion with BamHI in BamHI buffer for an additional hour at 37° C. The digested DNA was extracted using the phenol-chloroform method, followed by ethanol precipitation. The synthetic EGFP-coding region was ligated into an expression vector, which had a CMV promoter and a BGH 3′UTR and had been digested with the same restriction enzymes (SalI and XbaI) and purified by phenol-chloroform extraction, followed by ethanol precipitation. Cloning of the EGFP-DNA into the expression vector was performed using the following ligation reaction: 30 μg of digested vector DNA were mixed with 90 μg of digested EGFP-DNA in a 10 μl reaction containing T4 DNA ligase. The ligated products were used to transform DH5α competent E. coli cells followed by expansion of the resulting colonies in a bacterial culture medium. The recombinant DNA was extracted using a Qiagen plasmid purification kit (Qiagen, Germany). The sizes of the vectors harboring the inserts were verified using gel electrophoresis. The resultant expression vector with the modified UU/UA-reduced coding region along with a vector containing the wild type DNA were used for functional studies to confirm the expression of the encoded protein. HEK293 cells were grown at standard culture conditions (37° C., 5% CO2) in DMEM medium supplemented with 10% FBS and antibiotics (Invitrogen). 2.5×104 cells per well in 96-well plates were transfected with 100 ng of the vector with the modified UU/UA-reduced coding region of EGFP or the vector containing the wild-type EGFP-DNA. Transfections were performed in serum-free medium using Lipofectamine 2000 (Invitrogen) for 5 h, followed by replacing the medium with serum-supplemented medium. After approximately 24 or 48 hours, the plates were imaged and quantified using a BD high-content imager. Quantification was performed with a Proxcell imaging algorithm.
The data clearly shows that the use of the UU/UA-reduced coding region of EGFP allows a significantly (2.5 to 3-fold) higher expression of EGFP in eukaryotic cells than that of the wild type EGFP-DNA (FIG. 1).
(2) HEK293 cells were grown at standard culture conditions (37° C., 5% CO2) in DMEM medium supplemented with 10% FBS and antibiotics (Invitrogen). 2.5×104 cells per well in 96-well plates were transfected with 100 ng of the vector with the modified UU/UA-reduced coding region of EGFP or the vector containing the wild-type EGFP-DNA. The cells were also co-transfected with either an empty control vector (pcDNA 3.1) or a RNase L vector (pcDNA 3.1). Transfections were performed in serum-free medium using Lipofectamine 2000 (Invitrogen) for 5 h, followed by replacing the medium with serum-supplemented medium. After approximately 24 or 48 hours, the plates were imaged and quantified using a BD high-content imager. Quantification was performed with a Proxcell imaging algorithm.
The data shows that—in comparison/contrast to the wild type EGFP sequence—the use of the modified EGFP sequence resulted in higher expression, which was not constrained by the co-expression of RNase L (FIG. 2).
(3) Expression active PCR products were generated by using primers in which the forward (5′) primers were complementary to the beginning of the CMV promoter region or a sequence upstream of the CMV promoter, while the reverse (3′) primers were complementary to the BGH poly A site or a sequence downstream of this site. The PCR was carried out using a mixture of Taq and Pfu polymerase in a 100 μl reaction with the following cycle conditions: —95° C. for 12 min (to activate hot start polymerases), —32 cycles of: 94° C., 1 min; 52° C., 1 min; 72° C., 4 min, and a final extension at 72° C. for 7 min. The PCR products were purified using Qiagen PCR purification columns to eliminate the primers, small PCR products, buffer, and enzymes, and eluted in sterile water. HEK293 cells were grown at standard culture conditions (37° C., 5% CO2) in DMEM medium supplemented with 10% FBS and antibiotics (Invitrogen). 2.5×104 cells per well in 96-well plates were transfected with 100 ng of purified PCR products generated from the EGFP expression vector with the wild type or with the modified sequence. Transfections were performed in serum-free medium using Lipofectamine 2000 (Invitrogen) for 5 h, followed by replacing the medium with serum-supplemented medium. After approximately 24 or 48 hours, the plates were imaged and quantified using a BD high-content imager. Quantification was performed with a Proxcell imaging algorithm.
The data shows that PCR products harboring the UU/UA-reduced coding region of EGFP led to higher expression of EGFP (5 to 10-fold increase) than those harboring the wild type sequence (FIG. 3).
(4) Using the same methodology as described in (2) and (3), Hek293 cells were transfected with wild type or UU/UA-reduced firefly luciferase expression vector (“Superluciferase”, SEQ ID NO: 15). The luciferase activity levels were quantified by a luminometer. The data show that, within two independent experiments, there was an approximately 5- and approximately 100-fold difference (FIG. 4).
Likewise, Huh7 cells were transfected with different PCR products generated in accordance with the methodology outlined in (3) above, from the wild type or modified firefly luciferase expression vector (“Superluciferase”; SEQ ID NO: 15). The luciferase activity levels were, again, quantified by a luminometer. The data show that PCR products harbouring the UU/UA-reduced coding region of superluciferase led to a substantially higher expression of luciferase (20-100-fold increase) than those harbouring the wildtype sequence (FIG. 5). This demonstrates the method in accordance with the present invention works with a verity of variety of reporter proteins.
Likewise, Hek293 cells were transfected, using the same methodology as in (2), above, with a wild type or UU/UA-reduced hepatitis B surface (SEQ ID NO: 23) antigen expression vector. The expressed protein was quantified as mIU/ml. There was approximately a 4-fold difference (FIG. 6), but it is likely that this may be even higher in independent experiments.
This is an example that the method in accordance with the present invention also works with therapeutic proteins, antibodies and vaccines which have been modified, i.e. their coding sequence has been UU/UA-reduced, and this leads to a substantial increase in expression.
(5) In order to reflect the transcriptional changes and the subsequent effects on reported protein levels, protein-destabilizing amino acid regions that include a PEST sequence (=peptide sequence which is rich in proline, glutamic acid, serine and threonine) have been used to reduce the half-life of various reporter proteins. PEST sequences are associated with proteins that have a short intracellular half-life. Li et al. (J. Biol. Chem., 1998, 273, pp. 34970-34975) describe the use of a PEST sequence of MODC to destabilize the EGFP, and Leclerc et al. (Biotechniques, 2000, 29, pp. 590-591, pp. 594-596 used a PEST sequence to reduce the protein half-life of firefly luciferase.
Using such MODC-domain (mouse ornithine decarboxylase), more specifically, amino acids 422-461 of the degradation domain of the highly unstable MODC, the present inventor rendered a number of reporter genes unstable by fusing them with the afore-mentioned MODC domain. Moreover, the present inventor modified such fusions by reducing the number of UU/UA dinucleotides in both the reporter gene and the MODC domain in accordance with the present invention with respect to EGFP from Aequorea Victoria, Montastrea Cavernosa, Clavularia and Puntelina Plumate. The number of UU/UA dinucleotides was reduced both in the EGFP-part and the MODC part of the fusion.
The MODC domain was amplified from genomic DNA of mouse fibroblasts using specific primers that contain EcoRI and BamHI sites in the forward and reverse primer, respectively. The amplified cDNA was cloned in frame with the GFP coding region using the same restriction sites. Hek293 cells were transfected with destabilized GFPs as indicated in FIG. 7. The fluorescence intensity was quantitated by imaging apparatus and software. Compared to destabilized wildtype EGFP from Aequorea Victoria (i.e. the point of reference was wildtype EGFP, SEQ ID NO: 8, fused to wildtype MODC, SEQ ID NO: 29), there was a two-fold increase in fluorescence from modified Aequorea Victoria, an 8-fold increase from modified Montastrea Cavernosa green fluorescent protein, a 4-fold increase from modified Clavularia green fluorescent protein and a 7-fold increase from modified Puntelina Plumate green fluorescent protein (see FIG. 7). The term “modified” here means “UU/UA reduced and fused with MODC which itself has also been UU/UA reduced”.
Consequently, this shows that the present invention also works in situations where expression signals normally are weaker, and improves the fold-increase in expression in such situations.
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## Claims

1. A method for increasing the expression of a protein in a cell, said method comprising the step of reducing the number of RNase L cleavage sites in a nucleic acid sequence encoding said protein, and wherein the cell is a eukaryotic cell.

2. The method according to claim 1, wherein the number of RNase L cleavage sites is reduced by at least 10%.

3. The method according to claim 1, wherein said cleavage sites are UU and/or UA dinucleotides.

4. The method according to claim 1, wherein the step of reducing the number of RNase L cleavage sites reduces the number of said sites in a coding region of said nucleic acid sequence.

5. The method according to claim 4, wherein the step of reducing the number of RNase L cleavage sites in said nucleic acid sequence is performed without altering the amino acid sequence of said protein.

6. The method according to claim 5, wherein in the step of reducing the number of RNase L cleavage sites a codon comprising a UU and/or UA dinucleotide is exchanged for an alternative codon not comprising a UU and/or UA dinucleotide and coding for the same amino acid.

7. The method according to claim 5, wherein in the step of reducing the number of RNase L cleavage sites at least one codon of an adjacent pair of codons comprising a UU and/or UA dinucleotide is exchanged for an alternative codon coding for the same amino acid so that said adjacent pair of codons no longer comprises a UU and/or UA dinucleotide.

8. The method according to claim 6, wherein said alternative codon is a more frequently used codon in said cell.

9. The method according to claim 1, wherein the step of reducing the number of RNase L cleavage sites reduces said number in a non-coding region of said nucleic acid sequence.

10. The method according to claim 9, wherein said non-coding region is a 5′UTR, a 3′UTR, or an intron.

11. The method according to claim 9, wherein the step of reducing the number of RNase L cleavage sites is performed by mutation, deletion, or insertion of one or more nucleotides.

12. The method according to claim 1, further comprising the step of codon optimization prior to the step of reducing the number of RNase L cleavage sites.

13. The method according to claim 1, further comprising the step of transfecting said nucleic acid sequence encoding said protein into said cell in an expression active PCR product or contained in an expression vector after the step of reducing the number of RNase L cleavage sites.

14. The method according to claim 13, further comprising the step of translating said protein from said expression active PCR product or expression vector in said cell.

15. The method according to claim 1, wherein said protein is selected from the group consisting of reporter proteins, therapeutic proteins, antibodies, vaccines, membrane proteins, fusion proteins, blood plasma proteins, cytokines, interferons, growth factors, chemokines, and GPCRs.