Patent Publication Number: US-2019185865-A1

Title: Use of prokaryotic transcriptional activators as metabolite biosensors in eukaryotic cells

Description:
FIELD OF THE INVENTION 
     The present invention relates to the use of transcriptional activators from prokaryotic organisms for use in eukaryotic cells, such as yeast as sensors of intracellular and extracellular accumulation of a ligand or metabolite specifically activating this transcriptional activator in a eukaryote, such as yeast cell, such as a cell engineered to produce this ligand. The transcriptional activator controls a promoter upstream of a gene that may include e.g. a reporter gene that may be a fluorescence marker, such as luciferase or green fluorescent protein. 
     BACKGROUND OF THE INVENTION 
     Whole-cell biocatalysts have proven a tractable path towards sustainable production of bulk and fine chemicals. Yet, screening libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavour. For this reason the development of biosensors enabling real-time monitoring of product formation has gained significant attention. 
     Bio-based production of chemicals and fuels is an attractive avenue to reduce dependence on petroleum. For bio-based production, biocatalysts must often be genetically modified in order to increase product titers, rates and yields. However, the current efficiency of genome engineering methods and parts prospecting allow for unprecedented genotype diversity that vastly outstrips our ability to screen for best cell performance. 
     To meet this demand, bioengineers have started to develop genetically encoded devices and systems that enable screening and selection of better-performing biocatalysts in higher throughput. Genetic devices like oscillators, amplifiers and recorders, which have been developed based on fine-tuned relationships between input and output signals are promising tools for programming and controlling gene expression in living cells. These devices sense extra- or intracellular perturbations and actuate cellular decision-making processes akin to logic gates in electrical circuits. For instance, AND logic gates have been built using inducible expression of two split T7 RNA polymerase domains which control reporter gene expression when used in combination with a complimentary set of altered T7-specific promoters. From a diverse set of inputs, other molecular gating components like RNA aptamers, allosteric regulators and ligand-binding transcription factors have been engineered to control outputs for applications such as high-throughput screening, actuation on cellular metabolism, and evolution-based selection of optimal cell performance. 
     A key component in many of the reported devices is a ligand-inducible transcriptional regulator. Transcriptional regulators are powerful components finding many uses in genetic designs. Owing to their modular structure, transcriptional regulators have proven to be versatile platforms for genetically encoded Boolean logic functions. In particular, gene switches based on ligand-binding transcriptional repressors bind to genomic targets in the absence of their cognate ligand and thereby repress gene expression of the downstream gene(s), whereas binding between ligand and repressor causes the release of the repressor from the DNA and thereby a de-repression. In NOT gates like this, the simple steric hindrance of RNA polymerase progression, like in the case of the tetracyclin-responsive gene switch TetR, have for decades been used for conditional control of gene expression in both prokaryotic and eukaryotic chassis. Most importantly, transcriptional repressors and other artificial transcriptional regulators can be further engineered—including the addition of nuclear localization signals, destabilization domains and transcriptional activation regions—to repurpose conditional repressors into activators. Though conceptually intriguing and practically relevant, the repurposing and engineering of logic gates can suffer from the inherent need for extensive engineering. 
     Though most ligand-inducible genetic devices adopted for eukaryotes have historically been founded on transcriptional repressors, a hitherto untapped resource for use in genetic designs is ligand-inducible transcriptional activators. Remarkably, bacterial genomes encode a multitude of ligand-inducible activators amenable for integration into synthetic genetic devices. In bacteria, transcriptional activation takes place through (i) a promoter-centric or (ii) an RNA polymerase-centric mechanism. In the former case a transcriptional activator can bind to an operator site in a promoter thereby improving its ability to guide RNA polymerase to initiate transcription, whereas in the latter case activation relies on interactions with the RNA-polymerase itself such as when a housekeeping a factor is replaced by another a factor. Examples of prokaryotic transcriptional activators used for genetic designs in other non-native prokaryotic chassis include arabinose-inducible AraC and quorum sensing LuxR. However, so far no direct transplantation of prokaryotic ligand inducible transcriptional activators has been reported in eukaryotes. As the number of bulk and fine chemicals produced in eukaryote chassis continues to increase, there is an increasing need to be able to regulate these pathways using any and all available means, including the heretofore untapped prokaryotic ligand-inducible transcriptional activators. 
     OBJECT OF THE INVENTION 
     It is an object of embodiments of the invention to provide eukaryotic cells comprising bacterial transcriptional activator systems functioning in a eukaryotic chassis. Accordingly some genes within the eukaryotic cell are under influence of a bacterial transcriptional activator preferably working in a eukaryotic cell on the endogenous promoter of this eukaryotic cell. 
     It is an object of embodiments of the invention to provide eukaryotic cells that contain sensors, such as easily visible sensors of intracellular and extracellular accumulation of a ligand or metabolites being produced by this cell. 
     The inventors of the present invention have applied systematic engineering of multiple parameters to search for a general biosensor design based on small-molecule binding transcriptional activators from the prokaryote super-family of LysR-type transcriptional regulators (LTTRs). The present inventors have identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proofs-of principle they have applied the biosensors for in vivo screening of cells producing naringenin or cis, cis-muconic acid at different levels, and show that reporter gene output correlates with product accumulation. The transplantation of prokaryotic transcriptional activators into a eukaryotic chassis illustrates the potential of a hitherto untapped resource for engineering biosensors useful for biotechnological applications. 
     The present inventors have found a direct onboarding of a prokaryotic transcriptional activator as a biosensor for e.g. cis, cis-muconic acid (CCM) in budding yeast  Saccharomyces cerevisiae . Based on a multi-parametric engineering strategy the present inventors identified a functional design for the biosensor. Most importantly the design is applicable to a range of other LTTR-based biosensors founded on small-molecule induced transcriptional activators. As proofs-of-principle two of these biosensors were applied for real-time monitoring of bulk and fine chemical product accumulation in yeast cells engineered to produce CCM and naringenin, respectively. This constitutes the first successful direct transfer of prokaryotic transcriptional activators into a eukaryotic chassis to activate gene expression merely by placing the binding site of a transcriptional activator at a defined location in a reporter promoter and without reconfiguring any other motifs and domains. 
     Systematic engineering and meticulous characterization have for decades pushed forward the sequence-function understanding of genetic parts and interactions thereof. This has allowed the rational engineering of parts and genetic circuits useful for a range of applications within biotechnology. While most of the genetic devices originate from prokaryotes, transplantation into eukaryotes has been reported for a number bioswitches in order to construct orthogonal genetic devices to control a cellular response to a defined input. Specifically, genetic devices enabling the manipulation of transcription through the transplantation of prokaryote transcriptional repressors have inspired researchers, in their quest for tools to screen, select and actuate on cellular responses. In this study we have shown that ligand-inducible transcriptional activators from the largest family of transcriptional regulators found in prokaryotes, can be ported to eukaryotic chassis and used to measure the level of a small molecule inside the cell and activate transcription. The LTTR-based transcriptional activators function as is in yeast without any further engineering nor the co-expression of other molecular components (i.e. 6 factors). In fact, through a systematic engineering approach we provide a framework from which new ligand-binding transcriptional activators from the LTTR family can be designed through the simple swapping of a candidate LTTR operator sequence into the 209 bp_CYC1p truncated endogenous promoter at a defined position (T1)( FIG. 2 a - b   , Table 4, and Table 5). Also, and most importantly, we provide two successful proofs-of-principle for such biosensors to screen in vivo for the best-performing biocatalysts. 
     Compared to many of the studies using transcriptional repressors as biosensors in eukaryotes, the biosensor outputs based on ligand-inducible transcriptional activators presented in this study have lower dynamic ranges falling within one order of magnitude. This is in agreement with the observation from using BenM and FdeR as biosensors in  E. coli . This can pose a challenge for their applicability in genetic designs where a larger dynamic range is needed. However, we demonstrated in this study how these biosensors could be subjected to biosensor-based FACS for identification of biosensor designs with improved characteristics (ie. dynamic range), which may expand their applicability for metabolic engineering. For sure, we envision this to be exploitable for high-throughput screening of libraries of genetic designs for metabolites for which there exists no high-throughput screening assay or biosensor. 
     Apart from dynamic range, another key performance measure for biosensors, is their operational range. In our study we demonstrated how biosensors could be used in laboratory strains with limited engineering towards titer improvements, which at their best still are far from commercially relevant. Indeed, in diploid yeast, production of 559.3 mg/L CCM was recently reported, whereas an  E. coli - E. coli  co-cultivation study have reported the production of 2 g/L CCM. Though tolerance to low-pH fermentations should make yeast an economically feasible chassis for biobased production of dicarboxylic acids like CCM, the CCM biosensor design based on BenM may need to be adjusted or evolved as production hosts become better and the product titers gets higher. Additionally, the biosensor will need to be matched to the production kinetics of the individual strain or library of biocatalysts. 
     Nevertheless, the LTTR-based ligand-inducible transcriptional activators reported here are much-needed tools for optimizing biocatalysts that produce chemicals and fuels for which there exist no high-throughput screen or selection. This should spur interest in developing many other orthogonal logic gates based on LTTR members, which could serve as a vast and valuable reservoir for developing new ligand-inducible genetic circuits capable of high-throughput screening, reprogramming and growth-coupled strain selection for bio-based production of chemicals. Furthermore, as the mode-of-action of transcriptional activators (YES) differ from that of repressors (NOT), the future possibility for higher-order designs within cellular reprogramming can now be exploited in greater diversity. 
     SUMMARY OF THE INVENTION 
     It has been found by the present inventor(s) that transcriptional activators from prokaryotic organisms may work in a eukaryotic chassis by the positioning of the operator at a particular place within the eukaryotic promoter. The inventors found that this can be used e.g. for providing biosensors for intracellular and extracellular accumulation of a ligand or metabolites produced within a eukaryotic cell. 
     So, in a first aspect the present invention relates to a eukaryotic cell, such as a yeast cell comprising a bacterial transcriptional activator and a corresponding operator sequence positioned in a eukaryotic promoter, such as positioned within an endogenous promoter of said cell, which activator controls the expression of a gene from said eukaryotic promoter. 
     In a second aspect the present invention relates to a eukaryotic cell, such as a yeast cell comprising a bacterial transcriptional activator and a corresponding operator sequence, which activator controls the expression from a eukaryotic promoter, such as an endogenous promoter of the cell in response to a ligand specifically binding the transcriptional activator. 
     In a third aspect the present invention relates to a eukaryotic cell, such as a yeast cell comprising a bacterial transcriptional activator and a corresponding operator sequence positioned in a eukaryotic promoter, such as positioned within an endogenous promoter of the cell, which activator controls the expression of a gene from the eukaryotic promoter depending on the presence, such as dose dependent, of a ligand specifically binding the transcriptional activator 
     In a further aspect the present invention relates to the use of a prokaryotic transcriptional activator as a regulator of transcription in a eukaryotic cell, such as a yeast cell according to the invention; the transcriptional activator being activated by a ligand specifically binding the transcriptional activator to induce the expression of a protein product from a eukaryotic promoter of the cell; the promoter containing the operator sequence corresponding to the transcriptional activator. 
     In a further aspect the present invention relates to a the use of a prokaryotic transcriptional activator as a regulator of transcription in a eukaryotic cell, such as a yeast cell according to the invention; the transcriptional activator being activated depending on the presence, such as dose dependent, of a ligand specifically binding the transcriptional activator to induce the expression of a protein product from a eukaryotic promoter of said cell, the promoter containing the operator sequence corresponding to said transcriptional activator positioned within the promoter. 
     In a further aspect the present invention relates to a the use of a prokaryotic transcriptional activator as a metabolite biosensor for measuring the amount of a ligand extracellular of and/or produced by a eukaryotic cell, such as a yeast cell according to the invention, wherein the ligand specifically bind the transcriptional activator to induce expression of a reporter gene from a eukaryotic promoter of the cell, the promoter containing the operator sequence corresponding to the transcriptional activator. 
     In a further aspect the present invention relates to a method for measuring the amount of a ligand intracellular or extracellular of a eukaryotic cell, such as a yeast cell; said cell comprising a bacterial transcriptional activator and a corresponding operator sequence, which activator controls the expression of a reporter gene from a eukaryotic promoter of said cell in response to said ligand specifically binding said transcriptional activator; said promoter containing the operator sequence corresponding to said transcriptional activator; said method including the steps of 
     a) Cultivating a eukaryotic cell according to the invention;
 
b) Measuring the output from said promoter of said reporter gene;
 
c) Correlating said output from step b) with amount of said ligand.
 
     In some embodiments the ligand is not produced by the eukaryotic cell, but is present in a solution of the cultivation medium of the eukaryotic cell, or such as when used to report toxic waste in a soil. 
     In a further aspect, the present invention relates to a recombinant transcriptional activator with increased activity, such as BenM with mutations at any one or more of the positions H110R, F211V, and Y286N. 
    
    
     
       LEGENDS TO THE FIGURE 
         FIG. 1 . Onboarding the cis-cis-muconic acid (CCM) responsive prokaryotic transcriptional activator BenM in yeast. (a) Schematic outline of native and synthetic full-length (491 bp) CYC1 promoter variants with different BenO positioning and number (T1 and/or T2). The transcriptional activator BenM from  Acinetobacter  sp. ADP1 controls expression of GFP from the synthetic CYC1 promoter with BenM operator (BenO) integrated at position T1 and/or T2. CCM further induces BenM-dependent expression of GFP (b) Mean fluorescence intensity (MFI) values from flow cytometry measurements of GFP intensities in the presence or absence of BenM expressed from the constitutively active TEF1 promoter, and following 24 h of incubation in the presence or absence of 1.4 mM CCM. (c) Screening 84 yeast strains expressing all possible combinations of BenM expression levels (TDH3p, TEFp, RNR2p and REV1p) individually or in combination with native or engineered CYC1p reporter promoters of different lengths (491 bp, 272 bp, 249 bp and 209 bp), BenO positioning, and number (T1 and/or T2) by flow cytometry. Outputs are ordered according to GFP intensity in control medium. Dashed lines indicate background fluorescence as inferred from BenM expressing strains without GFP, and arrow indicate best-performing biosensor design. Genotypes and GFP expression levels of all 84 strains are listed in Tables 1 and 2, respectively. (d) Heat-maps showing fold change (FC) in GFP expression between CCM-induced and control cultures of 80 strains shown in (c). For (b) and (c) mean fluorescence intensities (MFI) are shown as mean±s.d. from three (n=3) biological replicate experiments. AU, arbitrary units. 
         FIG. 2 . High-throughput engineering and screening of BenM variants with improved CCM-inducibility. (a) Purified products from three rounds of error-prone PCR (epPCR) using the effector-binding domain (EBD) of BenM as template, were co-transformed into yeast together with a linearized centromeric plasmid, to allow for in vivo library reconstitution by gap repair and expression of wild-type BenM DNA binding domain (DBD) fused to approx. 40,000 variants of the EBD. Transformed yeast contained a chromosomal integration of 209 bp_CYC1p_BenO_T1 controlling the expression of GFP to allow for FACS-based screening of BenM variants with improved CCM-inducibility. (b) Representative flow cytometry histograms of fluorescence intensities obtained from a population of yeast cells expressing CCM sensor variants in control (grey) and CCM-inducer (light green) media. Control, CCM-induced and sorted (darker green) cell populations are normalized to mode for comparison. The proportions of cells within each histogram were calculated by FlowJo software as described in Methods (c) Isolated BenM variants were grown as clonal cultures, validated by flow cytometry and the EBDs of variants with significantly higher GFP expression under CCM-induced cultivation were sequenced. Mean fluorescence intensities (MFI) are shown as mean±s.d. from three (n=3) biological replicate experiments. AU, arbitrary units. (d) Ribbon representations of the EBD of BenM (PDB 2F7A) with the residue changes identified in BenM H110R, F211V, Y286N  highlighted in green. Bound CCM is highlighted as a magenta Van der Waals sphere. 
         FIG. 3 . Biosensor specificity and transcriptional orthogonality. (a) The specificity of the CCM biosensor was tested by addition of various dicarboxylic acids (1.4 mM) to the growth medium. GFP expression was measured by flow cytometry following 24 h of cultivation. (b) RNA sequencing FPKM (fragments per kilo base per million) are plotted for yeast cells stably expressing 209 bp_CYC1p_BenO_T1::GFP reporter construct and BenM H110R, F211V, Y286N  versus cells only expressing the reporter construct following 24 h of cultivation in medium supplemented with CCM. Purple area indicates 2-fold cut-off and red dots significantly differentially regulated genes as inferred from cuffdiff (&gt;2-fold, P&lt;0.05)(see also  FIG. 5 ). All data points are averaged from three (n=3) biological replicates. 
         FIG. 4 . Onboarding transcriptional activators from the LTTR family as biosensors in yeast. (a) Left: Schematic illustration of LTTR-mediated activation of GFP expression by binding to the cognate operator in position T1 of 209 bp_CYC1p. Right: The 209 bp_CYC1p_T1 reporter promoter design supports GFP expression when controlled by individual LTTR transcriptional activators expressed from either a weak (REV1p) or a strong (TDH3p) promoter. The y-axis shows fold induction in mean fluorescence intensity (MFI) in cells expressing individual LTTRs relative to cells not expressing the LTTRs. (b) Left: Schematic illustration on external application of individual ligands for induction of LTTR-mediated activation of GFP expression. Right: External application of individual ligands can induce LTTR-mediated activation of GFP expression. The y-axis shows fold induction in mean fluorescence intensity (MFI) for cells grown for 24 h in medium containing either cis, cis-muconic acid (CCM), naringenin (NAR), L-arginine (ARG), protocatechuic acid (PCA) or malonic acid (MAL) compared to cells growing in control medium. (c) Heatmap showing orthogonality of MdcR- and ArgP-mediated transcriptional regulation of GFP expression controlled by either reporter promoter 209 bp_CYC1p_MdcO_T1 or 209 bp_CYC1p_ArgO_T1 (Table 4). Color key shows mean fluorescence intensity (MFI) from three (n=3) biological replicate experiments. For (a) and (b), mean fluorescence intensity (MFI) values and their error bars are calculated as mean±s.d. from three (n=3) biological replicates. 
         FIG. 5 . Biosensor sensitivity and operational range. (a) The response functions of wild-type and engineered BenM H110R, F211V, Y286N  expressed in yeast from REV1p as measured by flow cytometry using various concentrations of CCM (24 h) and the 209 bp_CYC1p_BenO_T1 promoter controlling the expression of GFP. A yeast strain without BenM expressed is used as a control for background GFP fluorescence from the 209 bp_CYC1p_BenO_T1 promoter. (b) The response function measurement for the naringenin biosensor when FdeR is expressed from a weak (REV1p) or a strong (TDH3p) promoter using various concentrations of naringenin (24 h) and the 209 bp_CYC1p_FdeO_T1 reporter promoter controlling the expression of GFP. A yeast strain without FdeR expressed is used as a control for background GFP fluorescence from the 209 bp_CYC1p_FdeO_T1 promoter. For (a) and (b) mean fluorescence intensity (MFI) values and their error bars are calculated as mean±s.d. from three (n=3) biological replicate experiments 
         FIG. 6 . In vivo application of CCM and naringenin biosensors in yeast. (a) Schematic representation of the heterologous 3-step CCM production pathway for testing BenM as a biosensor for in vivo CCM production in yeast. Additionally, over-expression of Tkl1 was included together with balancing of the heterologous three-step pathway (PaAroZ, KpAroY and CaCatA) using single or multi-loci integration of AroY subunits B and C (Iso, isoform)(see Methods and Table 1). (b) Following 24 h of cultivation, CCM titers and MFIs were quantified and plotted for each strain. (c) Schematic representation of heterologous 5-step naringenin production pathway adopted from Naesby et al. For the hydroxylation of cinnamate to coumarate a fusion protein of AtC4H and AtATR2 was used. For testing FdeR as a biosensor for in vivo naringenin production in yeast, mean fluorescence intensity (MFI) in three different strains engineered with one copy of the 5-step naringenin production pathway (EVR1) or with one (EVR2) or two (EVR3) additional integrations of bottleneck enzymes were compared to a control strain (EVR0, ctrl) without the production pathway. Following 48 h of cultivation, naringenin titers and MFIs were quantified and plotted. For both (b) and (d) data are average of three biological replicates. Mean fluorescence intensity (MFI) values and metabolite quantifications are presented as means±s.d. from three (n=3) biological replicate experiments. 
         FIG. 7  BenM regulation of the ben operon during benzoate catabolism in  Acinetobacter  sp. ADP1 is feed-back induced by the intermediate catabolite cis,cis-muconic acid (CCM). Upon detection of CCM from the aromatic acid catabolism, the constitutively DNA-bound BenM tetramer undergoes a conformational change facilitating the accessibility of RNA polymerase and active transcription of the ben operon (Bundy, B. M., Proc. Natl. Acad. Sci. U.S.A 99, 7693-8 (2002). Sequence of the BenM operator (BenO). The three potential binding sites for BenM are highlighted in blue with site 1 displaying dyad symmetry exactly matching the consensus sequence of LysR-type regulators (Collier, L. S., 3. Bacteriol. 180, 2493-501 (1998)). 
         FIG. 8 ( a )  Sequence outline of the full-length CYC1 promoter with native upstream activating sequences (UASs) shown in blue and operator sites for TATA-binding proteins shown in red. Sites for positioning of BenM operators are marked with black triangles (T1 and T2) and sites for truncations (272 bp, 249 bp and 209 bp) marked with dashed vertical lines. USER cloning site and Kozak sequence is italicized upstream the open reading frame of yeast-enhanced GFP (bold). (b) Sequence of the FdeR, PcaQ, ArgP and McdR operators used to swap into position T1 of the 209 bp_CYC1p shown in (a) (Siedler, S., Metab. Eng. 21, 2-8 (2014) and Maclean, A. M., Microbiology 157, 2522-33 (2011). 
         FIG. 9 ( a )  Uptake of cis,cis-muconic acid (CCM) at pH 4.5 by  S. cerevisiae  cells following 24 h of growth. (b) Representative growth curves of yeast cells in liquid medium containing different concentrations of CCM. OD values were determined at 1-h intervals over 25-h period. For both (a) and (b) data display means±s.d. from three (n=3) biological replicate cultivations. 
         FIG. 10  Screening 84 yeast strains expressing all possible combinations of BenM expression levels (TDH3p, TEFp, RNR2p and REV1p) individually or in combination with native or engineered CYC1p reporter promoters of different lengths (491 bp, 272 bp, 249 bp and 209 bp), BenO positioning, and number (T1 and/or T2) by flow cytometry after 24 h of growth in control medium or medium supplemented with 1.4 mM CCM. Outputs are ordered according to GFP intensity in control medium. Dashed lines indicate background fluorescence as inferred from strains expressing only BenM (no reporter). Genotypes and GFP expression levels of all 84 strains can be found in Tables 1 and 2, respectively. Mean fluorescence intensity (MFI) values and their error bars are calculated as mean±s.d. from three (n=3) biological replicate experiments. AU, arbitrary units. 
         FIG. 11 ( a )  A box and whisker plot showing the mean value, 1 and 99 percentiles for three (n=3) biological replicate RNA sequencing experiments. Outliers are depicted as black dots. The GFP is highlighted in green. RNA was collected following 24 h of cultivation in mineral medium pH 4.5 with 1.4 mM CCM. The fold change in FPKM is displayed as a log 2 normalized value for all expressed genes. (b) A fold change histogram of FPKM showing the fold changes (log 2) in gene expression from  FIG. 2 c    plotting strain including BenMH110R, F211V, Y286N (MeLS0284) over the strain without BenMH110R, F211V, Y286N (MeLS0138). GFP is indicated with an arrow. The data are representative of three (n=3) biological replicates. 
         FIG. 12  Endogenous response function of the CCM and naringenin biosensors. (a) Cultivation medium was analyzed for CCM concentration by LC-MS and flow cytometry performed for GFP intensity measurements of six different CCM producing strains compared to a reference CCM null background strain (see Table 1) following 24 h and 72 h cultivations. (b) Average titers for the six CCM-producing strains at 24 h and 72 h of cultivation, compared to the reference strain. (c) Cultivation medium was analyzed for naringenin concentration by UPLC and flow cytometry performed for GFP intensity measurements of three different naringenin producing strains compared to a reference naringenin null background strain following 24 and 48 h cultivations. (d) Average titers for the three naringenin-producing strains at 24 h and 48 h of cultivation, compared to the reference strain. For (a-d) data are presented as means±s.d. from three (n=3) biological replicate experiments. Table 1 lists all strain genotypes. 
         FIG. 13 . BenM activates reporter expression in CHO. CHO cells were transfected with a plasmid with the BenO-containing human cytomegalovirus (CMV) promoter controlling the expression of GFP as well as an empty vector (− BenM) or a vector expressing BenM (+ BenM). Total GFP expression was measured after 24 h, and normalized by total RFP expression. Average and standard deviation are based on three biological replicates. *; p&lt;0.05 (t-test). 
         FIG. 14 . Screening 17 yeast strains expressing BenM from the REV1p in combination with CYC1p reporter promoter of 209 bp with BenO placed at different positions upstream of TATA1. The fold induction (mean±s.d.) was calculated by dividing mean fluorescence intensity (MFI) in medium with 1.4 mM CCM by the MFI in control medium as measured by flow cytometry after 24 h of growth for three biological replicates. 
     
    
    
     DETAILED DISCLOSURE OF THE INVENTION 
     Definitions 
     The term “eukaryotic cell” is used herein in its normal sense. The term includes any animal, mammalian, fungi, yeast, insect and algae cell. In some specific embodiments the eukaryotic cell is a yeast cell. 
     The term “yeast cell” refers to the single-celled microorganisms classified as members of the fungus kingdom. The term includes but is not limited to cells of a genus selected from the group consisting of  Kluyveromyces, Saccharomyces  and  Hanensula , such as a yeast cell selected from the group consisting of  Saccharomyces cerevisiae  and  Saccharomyces boulardii.    
     The term “bacterial transcriptional activator” as used herein refers to a protein, such as any know protein naturally derived from a bacterium (a transcription factor) that increases gene transcription of a gene or set of genes in this bacterium. It is to be understood that a bacterial transcriptional activator and its corresponding operator sequence (and whether it is derived from a prokaryote genome and not found in a eukaryotes motif) will be easily identified by a simple sequence search for the person skilled in the art. Essentially, for a transcriptional regulator to be defined as a prokaryotic transcriptional activators by the person skilled in the art it must adhere to all the following points: 
     a) The gene encoding the protein sequence is found natively in a prokaryotic genome.
 
b) When using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi) for comparing nucleotide or protein sequences to sequence databases, the query sequence aligns more to sequences of prokaryote origin in terms evolutionary relationships than to sequences of eukaryote origin.
 
c) In its native context of a prokaryotic genome, deletion of the gene encoding the protein sequence of the transcriptional regulator will cause lower or no change in expression of its target gene.
 
d) In its native context of a prokaryotic genome, over-expression of the gene encoding the protein sequence of the transcriptional regulator will not cause lower expression of its target gene, as is the case for transcriptional repressors.
 
e) The gene encoding the protein sequence is categorized functionally as an activator in the RegPrecise database (http://regprecise.lbl.gov); a database of curated genomic inferences of transcriptional regulatory interactions in prokaryotes.
 
     In some embodiments the bacterial transcriptional activator is within the prokaryote super-family of LysR-type transcriptional regulators (LTTRs). 
     In some embodiment the bacterial transcriptional activator as used herein is selected from the list of tables 6 and 7. 
     In some embodiments the term refers to an intact transcriptional activator containing both an activation domain and a DNA-binding domain. 
     The term “corresponding operator sequence” as used herein refers to the DNA sequence that binds a specific bacterial transcriptional activator in order to make the bacterial transcriptional activator take effect. The operator sequence is placed within the promoter on which the activator works. The corresponding operator sequence is identified as part of the DNA sequence of a prokaryote promoter sequence, which is under the regulation of the bacterial transcriptional activator. 
     The term “eukaryotic promoter” as used herein refers to a region of DNA derived from or within a eukaryotic cell that initiates transcription of a particular gene downstream of this promoter. 
     The term “endogenous promoter” as used herein refers to a promoter that normally is present in the cell in use. 
     The term “ligand specifically binding a transcriptional activator” as used herein refers to a ligand, which specifically binds to a particular transcriptional activator to control the functioning of the activator in a system of a so-called ligand-inducible transcriptional regulator. 
     The term “exogenous” refers to a gene that originates outside of the organism of the specific cell being used. 
     In some embodiments, the eukaryotic cell contains a reporter gene, preferably in operative linkage with the eukaryotic promoter responsive to the bacterial transcriptional activator. Exemplary reporter genes include enzymes, such as luciferase, phosphatase, or p-galactosidase which can produce a spectrometrically active label, e. g., changes in color, fluorescence or luminescence. In some embodiments the reporter gene encodes a gene product selected from the group consisting of luciferase, green fluorescent protein, p-lactamase chloramphenicol acetyl transferase, ss-galactosidase, secreted alkaline phosphatase, p-lactamase, p-glucuronidase, alkaline phosphatase, blue fluorescent protein, and chloramphenicol acetyl transferase. 
     “Upstream activating sequences” as used herein refers to cis-acting elements of a eukaryotic promoter that modulate the rate of initiation of transcription well known to the person skilled in the art. Specific sequence and number of subsites or regions is specific for the promoter being used. 
     Specific Embodiments of the Invention 
     As described above the present invention relates to a eukaryotic cell, such as a yeast cell comprising a ligand-binding bacterial transcriptional activator and a corresponding operator sequence positioned in a eukaryotic promoter, such as positioned within an endogenous promoter of the cell, which activator controls the expression of a gene from the eukaryotic promoter. 
     In some embodiments the expression of a gene from said eukaryotic promoter is depending on the presence, such as dose dependent, of a ligand specifically binding the transcriptional activator. 
     In some embodiments the cell comprises a gene encoding the expression of the ligand, one or more genes encoding a pathway of enzymes synthesizing the ligand, and/or a gene encoding a compound that is metabolized into the ligand. In some embodiment such a gene is expressed from the eukaryotic promoter. 
     In some embodiments the cell comprises an exogenous reporter gene, and/or one or more further regulatory gene, such as a gene encoding antibiotic resistance. In some embodiment such a gene is expressed from the eukaryotic promoter. 
     In some embodiments the reporter gene provides for fluorescence output, such as a gene encoding green fluorescent protein, blue fluorescent protein or luciferase. 
     In some embodiments the one or more the genes independently selected from the gene encoding the expression of the ligand, one or more genes encoding a pathway of enzymes synthesizing the ligand, a gene encoding a compound that is metabolized into the ligand, an exogenous reporter gene, and one or more further regulatory gene; is under the control and/or is activated by the eukaryotic promoter. 
     In some embodiments the transcriptional activator is selected from any one selected from table 6, such as any one selected from BenM, FdeR, MdcR, and ArgP. 
     In some embodiments the ligand and transcriptional activator is selected from muconic acid and BenM; Naringenin and FdeR; Malonate and MdcR, and L-arginin and ArgP. 
     In some embodiments the cell is a yeast cell, such as  Saccharomyces cerevisiae.    
     In some embodiments the cell is a mammalian cell, such as a Chinese hamster ovary cell. 
     In some embodiments the promoter is a full length promoter, or a truncated version with upstream activating sequences, such as UAS1 and UAS2 of the CYC promoter, removed. 
     In some embodiments the promoter is a yeast promoter, such as the full length CYC1 promoter or CYC1 with upstream activating sequences (UAS1 and UAS2) removed. 
     In some embodiments the promoter is a mammalian promoter, such as the full length CMV promoter. 
     In some embodiments the transcriptional activator work through a promoter-centric mechanism, wherein the transcriptional activator bind to an operator site in the promoter thereby improving its ability to guide RNA polymerase to initiate transcription. 
     In some embodiments the transcriptional activator does not require binding to any other regulatory subunits and/or which cell is without any further engineering or the co-expression of other molecular components regulating the transcriptional activator. 
     In some embodiments the transcriptional activator does not require binding to any other regulatory subunits apart from its specific ligand and/or which cell is without any further engineering or the co-expression of other molecular components regulating said transcriptional activator. 
     In some embodiments the operator sequence is specific for the transcriptional activator within the promoter. 
     In some embodiments the operator sequence is positioned immediately upstream of the TATA box, such as a TATA box 1, such as TATA-1β, such as anywhere between 6-15 bp, such as anywhere between 6-14 bp, such as anywhere between 6-13 bp, such as anywhere between 6-12 bp, such as anywhere between 6-11 bp, such as anywhere between 6-10 bp, such as anywhere between 6-9 bp, such as anywhere between 6-8 bp, such as anywhere between 6-7 bp, such as 6 bp upstream of said TATA box of said eukaryotic promoter. 
     In some embodiments the operator sequence is positioned immediately upstream of one of the two TATA boxes—TATA-1β, such as anywhere between 6-15 bp upstream of TATA box 1, such as anywhere between 6-14 bp upstream of TATA box 1, such as anywhere between 6-13 bp upstream of TATA box 1, such as anywhere between 6-12 bp upstream of TATA box 1, such as anywhere between 6-11 bp upstream of TATA box 1, such as anywhere between 6-10 bp upstream of TATA box 1, such as anywhere between 6-9 bp upstream of TATA box 1, such as anywhere between 6-8 bp upstream of TATA box 1, such as anywhere between 6-7 bp upstream of TATA box 1, such as 6 bp upstream of TATA box 1. 
     In some embodiments the operator sequence is positioned immediately 6 bp upstream of the TATA box—TATA-1β, such as anywhere between 6-15 bp upstream of TATA box 1, such as anywhere between 6-14 bp upstream of TATA box 1, such as anywhere between 6-13 bp upstream of TATA box 1, such as anywhere between 6-12 bp upstream of TATA box 1, such as anywhere between 6-11 bp upstream of TATA box 1, such as anywhere between 6-10 bp upstream of TATA box 1, such as anywhere between 6-9 bp upstream of TATA box 1, such as anywhere between 6-8 bp upstream of TATA box 1, such as anywhere between 6-7 bp upstream of TATA box 1, such as 6 bp upstream of TATA box 1. 
     In some embodiments the transcriptional activator belongs to the prokaryote super-family of LysR-type transcriptional regulators (LTTRs). 
     In some embodiments the operator is an LTTR operator sequence selected from BenO, FdeO, MdcO, and ArgO. 
     In some embodiments the transcriptional activator is co-expressed in the cell, such as from a promoter selected from TEF1, REV1, RNR2 and TDH3. 
     In some embodiments the transcriptional activator is a functional variant with increased activity, such as BenMH110R, F211V, Y286N. 
     Example 1 
     Strains, chemicals and media.  Saccharomyces cerevisiae  CEN.PK102-5B (MATa ura3-52 his3Δ1 leu2-3/112 MAL2-8 c  SUC2), CEN.PK113-5A (MATa, trp1 his3Δ1 leu2-3/112 MAL2-8 c  SUC2) and CEN.PK113-7D (wild type, MATa MAL2-8 c  SUC2) strains were obtained from Peter Kotter (Johann Wolfgang Goethe-University Frankfurt, Germany). In principal any other yeast strains may be used, such as one obtained from public repository EuroScarf. EasyClone plasmids used in this work are described in Jensen, N. B. et al. EasyClone: method for iterative chromosomal integration of multiple genes in  Saccharomyces cerevisiae . FEMS Yeast Res. 14, 238-48 (2014).  Escherichia coli  strain DH5a was used as a host for cloning and plasmid propagation. The chemicals and Pfu TURBO DNA polymerase were commercially obtained (Sigma-Aldrich and Agilent Technologies Inc., respectively). All acids used were &gt;97% in purity.  S. cerevisiae  cells were grown at 30° C. in synthetic complete medium as well as drop-out media and agar plates were prepared using pre-mixed drop-out powders (Sigma-Aldrich). Mineral medium was freshly prepared as described previously. For all media containing diacids, 1.4 mM of the individual diacids were dissolved in mineral medium and pH adjusted to 4.5 before sterile filtration. For CCM several dilutions were made to examine the performance of the CCM biosensor. For naringenin, mineral medium was supplemented with 0, 0.05, 0.10 or 0.20 mM naringenin, dissolved in ethanol, the final ethanol concentration for each medium was 2% (v/v), and the final pH of the medium was adjusted to 6.0.  E. coli  cells were grown at 37° C. in Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin. 
     Synthetic Genes and Oligonucleotides. 
     Oligonucleotides and synthetic genes were commercially synthesized (Integrated DNA Technologies, Inc. and Thermo Fisher Scientific Inc., respectively). Sequences of synthetic genes and oligonucleotides can be found in Tables 4 and 5, respectively. 
     Plasmids, Strains and Library Construction. 
     Except  Arabidopsis thaliana  At4CL-2 (NM 113019.3) and  Saccharomyces cerevisiae  ScTKL1 (NM_001184171.1), all genes encoding  Klebsiella pneumoniae  AroY.B (AAY57854.1), AroY.Ciso (BAH20873.1), AroY.D (AAY57856.1),  Candida albicans  CatA (XP_722784.1),  Podospora anserina  AroZ (XP_001905369.1),  Acinetobacter  sp ADP BenM (AAC46441.1),  Arabidopsis thaliana  AtC4H (NM_128601.2),  Arabidopsis thaliana  AtATR2 (NM_179141.2),  Arabidopsis thaliana  AtPAL2 (NM_115186.3),  Petunia hybrida  PhCHI (X14589),  Hypericum androsaemum  HaCHS (AF315345),  Schizosaccharomyces pombe  MAE1 (NM_001020205.2),  Sinorhizobium meliloti  PcaQ (NC_003078.1),  Escherichia coli  ArgP (NC_000913.3),  Klebsiella pneumonia  MdcR (U14004), and  Herbaspirillum seropedicae  SmR1 FdeR (Hsero_1002, UniProtKB-D8J0W4_HERSS) were codon-optimized for expression in yeast (see Table 4 for full sequences). All gene fragments and correct overhangs for USER-cloning were amplified by PCR using oligonucleotides listed and described in Table 5. Unless otherwise stated the amplified products were USER cloned into EasyClone integrative plasmids Jensen, N. B. et al. (2014), and confirmed by sequencing. 
     The list of the constructed plasmids can be found in Table 3. Transformation of yeast cells was carried out by the lithium acetate method Gietz, R. D. &amp; Schiestl, R. H. Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 38-41 (2007), and strains selected on synthetic drop-out medium (Sigma-Aldrich), selecting for appropriate markers. For selection of strain carrying KanMX and HypMX, the media was supplemented with 200 μg/mL G418 sulphate and 200 μg/mL hygromycin B, respectively. Transformants were genotyped using oligonucleotides described in Table 5. The resulting strains are listed in Table 1. 
     To establish the CCM producing strains, we expressed the dehydroshikimate DHS dehydratase from  P. anserina  (PaAroZ), the PCA decarboxylase genes from  K. pneumoniae  (KpAroY.B, KpAroY.Ciso, KpAroY.D), and the catechol 1, 2 dioxygenase CDO from  Candida albicans  (CaCatA) in  S. cerevisiae . It has been reported that the conversion of PCA to catechol by PCA decarboxylase is a limiting step. For this reason we expressed the KpAroY.B and KpAroY.Ciso genes in either single or multiple genomic integrations to create a small library of CCM production strains. In addition, Tkl1 was overexpressed in order to improve the precursor supply. 
     To establish a naringenin producing strain we integrated the full pathway containing the phenylalanine ammonium lyase from  Arabidopsis thaliana  (AtPAL-2), the fusion of cinnamate 4-hydroxylase from  Arabidopsis thaliana  and NADPH-cytochrome P450 reductase from  Arabidopsis thaliana  (AtC4H:L5:AtATR2), the 4-coumarate-CoA ligase 2 from  Arabidopsis thaliana  (At4CL-2), the naringenin-chalcone synthase from  Hypericum  androsaemum (HaCHS), and the chalcone isomerase from  Petunia hybrida  (PhCHI) to make strain EVR1 (table 1). Strains EVR2 and EVR3 contained one and two additional integrations of bottleneck enzymes (AtPAL-2 and HaCHS for EVR2; AtPAL-2, HaCHS, and AtC4H:L5:AtATR2 for EVR3)(table 1). 
     Mutagenesis of BenM and Library Preparation. 
     For optimization of the CCM inducibility of BenM, purified products from three consecutive rounds of error-prone PCR (epPCR) of the effector-binding domain (EBD, residues 90-304) of BenM, were co-transformed into yeast together with linearized centromeric plasmid according to Eckert-Boulet et al. (Eckert-Boulet, N., Pedersen, M. L., Krogh, B. O. &amp; Lisby, M. Optimization of ordered plasmid assembly by gap repair in  Saccharomyces cerevisiae . Yeast 29, 323-34 (2012)), to allow for in vivo gap repair and library reconstitution of wild-type BenM DNA binding domain (DBD) fused to EBD variants expressed from REV1p. For epPCR we used the GeneMorph II kit according to manufacturer&#39;s description (Agilent Technologies). Transformed yeast contained a chromosomal integration of the 209 bp_CYC1p_BenO_T1 promoter controlling the expressing of GFP at EasyClone site 4 on chromosome XII (Mikkelsen, M. D. et al. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab. Eng. 14, 104-11 (2012)), to allow for FACS-based screening of improved CCM-inducible BenM variants. 
     Metabolite Quantification Using HPLC and UPLC-MS. 
     The CCM production strains were cultivated in 24-deep well plate with air-penetrable lids (EnzyScreen) to test for the production of CCM. Colonies from the individual strain were inoculated in 1 mL synthetic drop-out medium (Sigma-Aldrich), selecting for URA, HIS and LEU markers, and grown at 30° C. with 250 rpm agitation at 5 cm orbit cast for 24 h. 300 μL of the overnight cultures were used to inoculate 3 mL mineral medium (pH 4.5) in 24-deep well plate and incubated for 24-72 h at the same conditions as above. Experiments were performed in triplicates. The culture broth was centrifuged 3,500×rpm and the supernatant analyzed for CCM concentration using HPLC. For this purpose, samples were analyzed for 45 min using Aminex HPX-87H ion exclusion column with a 1 mM H 2 SO 4  flow of 0.6 mL/min. The temperature of the column was 60° C. Refractive index and UV detectors (Dionex) were used for detection of CCM (250 nm). CCM concentrations were quantified by comparison with the spectrum of the standards. For the naringenin production strains 300 μl culture broth was extracted with 300 μl MeOH in a 10-minute incubation (300×rpm, 5 cm amplitude, 30° C.) in a 96 square deep-well microtiter plate (Greiner Masterblock, 96 Well, 2 ml, P, V-bottom) and subsequently clarified by centrifugation at 4000×g for 5 min. Clarified broth extract was subsequently diluted four times with 50% MeOH and 2 μl was injected on a Acquity UPLC system (Waters) coupled to a Acquity TQ mass detector (Waters). Separation of the compounds was achieved on a Acquity UPLC® BEH C18 column (Waters, 1.7 μm, 2.1 mm×50 mm), kept at 55° C. Mobile phases A and B were water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. A flow of 0.6 ml/min was used. The gradient profile was as follow: 0.3 min constant at 10% B, a linear gradient from 10% B to 25% B in 3.7 min, a second linear gradient from 25% B to 100% B in 1 min, a wash for 1 min at 100% B and back to the initial condition of 10% B for 0.6 min. The mass analyzer was equipped with an electrospray (ESI) source and operated in negative mode. Capillary voltage was 3.0 kV; the source was kept at 150° C. and the desolvation temperature was 350° C.; desolvation and cone gas flow were 500 L/h and 50 L/h respectively. EM-Hr ions of naringenin (271 m/z) was tracked in SIR mode. Naringenin was quantified using a quadratic calibration curve with authentic standards ranging from 0.039 mg/I to 20 mg/I using QuanLynx software (Waters). 
     Transport Assays. 
     Overnight grown CEN.PK113-5A cells were diluted (OD 600 =0.1) into SC-HIS-LEU medium with or without 1.4 mM (200 mg/L) CCM. Media samples were taken at both 0 h and 24 h, while samples for measuring cellular lysates (10 8  cells) were harvested at 24 h. For quantification of CCM by LC-MS, cultures were harvested by centrifugation. For extracellular CCM quantification the supernatant was centrifuged twice and filtered (0.2 μm) before analysis. For intracellular CCM quantification harvested pellets were washed twice in ice-cold isotonic saline solution (0.9% NaCl) and centrifuged at 5,000×g before cells were extracted in an aqueous 0.1% formic acid solution and sonicated for 15 min. Following this, samples were centrifuged at 13,000×g and supernatants filtered (0.2 μm) prior to analysis. LC-MS data was collected on EVOQ EliteTriple Quadrupole Mass Spectrometer system coupled with an Advance UHPLC (Bruker). Samples were held at 4° C. during the analysis. A 1 μL sample was injected onto a ACQUITY HSS T3 C18 UHPLC column (Waters), with a 1.8 μm particle size and 2.1×100 mm dimensioning. The column was held at a temperature of 30° C. The solvent system used was 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic (mobile phase B). The flow rate was 0.400 ml/min with an initial solvent composition of 100% mobile phase A held until 0.50 min, then changed until it reached % A=5.0 and % B=95.0 at 1.00 min. This was held until 1.79 min when the solvent was returned to the initial conditions and the column was re-equilibrated until 4.00 min. The eluent was sprayed into the heated ESI probe of the MS which was held at 250° C. and a voltage of 2500 V. Sheath/nebulizer/cone gas flow rate of 50/50/20 units and cone temp was 350° C. Two transitions were chosen in negative Multiple Reaction Monitoring (MRM) mode for quantification of CCM: m/z 141.70-96.80 (quantification transition) and m/z 141.70-53.1 (confirmation transition). Triplicate measurements were made for all samples. 
     Fluorescence Activated Cell Sorting 
     A two-step method was used to sort for BenM variants that specifically induce in the presence of CCM. Cells (10× library size, approx. 400,000 cells) were inoculated in mineral medium without inducer and incubated for 24 h at 30° C., diluted into PBS, and then GFP intensity of individual cells was measured using a BD Biosciences Aria (Becton Dickinson) with a blue laser (488 nm) by applying tight gates on the FSC and SSC channels. Only cells displaying auto-fluorescence intensities were sorted in order to limit auto-activating BenM variants. Sorted cells were recovered in mineral medium, followed by subculturing (1:100) into mineral medium containing 1.4 mM (200 mg/L) cis,cis-muconic acid. The cells were grown for 24 h at 30° C., washed and subjected to a second round of FACS. Cells exhibiting high levels of GFP (top 1%) were sorted, recovered in mineral medium and plated for single colonies on SC-HIS-LEU media. Individual clones were subsequently validated using flow cytometry. 
     Flow Cytometry Measurements and Data Analysis. 
     Cells grown for 24 h in control (mineral medium, pH 4.5) or inducing medium (mineral medium pH 4.5+1.4 mM CCM, 1.4 mM protocatechuic acid, 10 mM malonic acid, 0.2 mM naringenin, or 50 mM L-arginine) were diluted into PBS to arrest cell growth. Cells were then analyzed by flow cytometry using a Fortessa flow cytometer (Becton Dickinson) with a blue laser (488 nm), for validation of single strains. For each strain 10,000 single-cell events were recorded. Events were analyzed using FlowJo software (TreeStar Inc.). The fluorescence arithmetic mean of the gated cell population was calculated, and the fold-change determined by dividing the mean fluorescence of the induced (ON) state with the mean fluorescence of the control (OFF) state. For flow cytometry for CCM and naringenin producing cells we tested mean fluorescence intensities from 10,000 cells pr. strain following 72 and 48 h, respectively. The data represent the average of three (n=3) biological replicates and error bars correspond to the standard deviation between these measurements. 
     Transcriptome Analysis. 
     To study the impact of ligand-induced BenM on genome-wide gene expression, triplicate cultures of strains MeLS0138 and MeLS0284 were grown for 24 h at 30° C. in 50 ml mineral medium pH 4.5 with 1.4 mM CCM. Following this, total RNA was extracted essentially as previously described (Kildegaard, K. R. et al. Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance. Metab. Eng. 26, 57-66 (2014)). Briefly, 15 ml samples of the six cultures were harvested into a pre-chilled 50 ml tube with crushed ice and then immediately centrifuged at 4° C., 4000×rpm for 5 min. Subsequently, the pellets were resuspended in 2 ml of RNAlater® Solution (Ambion, Life Technologies) and incubated on ice for 1 h. Next, cells were pelleted by centrifugation (12,000×rpm for 10 s) and transferred to liquid nitrogen, and stored at −80° C. until further analysis. Total RNA extraction was performed using RNeasy® Mini Kit (QIAGEN). For this purpose, samples were thawed on ice, and 600 μl of buffer RLT containing 1% (v/v) 8-mercaptoethanol was added directly to the cells, before being transferred into a 2 ml extraction tube containing 500 μl glass beads and disrupted using the PRECELLYS®24 (Bertin Technologies) for 2×20 s at 6500×rpm. The cell mixture was pelleted and supernatant transferred to a new tube. Total RNA was purified according to the manufacturing&#39;s protocol, and genomic DNA removed using Turbo DNA-free™ Kit (Ambion). The quantity and quality of the RNA samples were measured using Qubit 2.0 Fluorometer using the Qubit RNA BR Assay (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit (Agilent Technologies), respectively. For sequencing we used 3 μg of total RNA as input for TruSeq® Stranded mRNA Sample Preparation kit prior to sequencing on the MiSeq System using MiSeq Reagent Kit v3 150 cycles at a 2×75 bp read length configuration (Illumina) obtaining 38 M reads. 
     Bioinformatic Resources. 
     Two-dimensional heatmap plots were generated using the plot3D package and the R GUI. For ribbon structure representation of CCM-binding domain of BenM H110,F211V,Y286N  the UCSF Chimera software was used (Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. 3. Comput. Chem. 25, 1605-12 (2004)). For RNA-seq data analysis, TopHat (2.0.13) and Cufflinks (2.2.1) suite was employed as previously described (Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562-78 (2012)). Expression levels (Fragments Per Kilobase of exon per Million fragments mapped: FPKM) from three (n=3) biological replicates of the conditions tested are processed with cuffdiff to obtain fold change differences and to perform statistical testing. A q-value cutoff of &lt;0.05 was used to identify genes that have significant differential expression. Additionally, a &gt;2-fold cut-off selection criterion was applied. Reference genome and annotations for CEN.PK113-7D strain were retrieved from  Saccharomyces  Genome Database (SGD; http://www.yeastgenome.org/). Genes with FPKM=0 for any replicate were removed from consideration. 
     Database for RNA-Seq Data. 
     RNA-seq data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-4836 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4836). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Strain names and genotypes of all strains generated in this study. Strain names for those shown in 
               
               
                 FIG. 1c are ordered according to basal activity (see also Supplementary Table 2) 
               
            
           
           
               
               
               
            
               
                 Strain 
                 Yeast Integrative 
                   
               
               
                 name 
                 Plasmid (parent strain) 
                 Genotype 
               
               
                   
               
               
                 CEN.PK113- 
                 — 
                 mat a URA3 HIS3 LEU2 TRP1 
               
               
                 7D 
               
               
                 CEN.PK102- 
                 — 
                 mat a ura3 his3 leu2 
               
               
                 5B 
               
               
                 Cen.PK113- 
                 — 
                 mat a his3 leu2 trp1 
               
               
                 5A 
               
               
                 MeLS0081 
                 pMeLS0045 + pCfB0262 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0153 
                 pCfB0262 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0079 
                 pMeLS0044 + pCfB0262 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0152 
                 pCfB0262 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0131 
                 pMeLS0077 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 491bp_CYC1p::yEGFP-SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0132 
                 pMeLS0078 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 272bp_CYC1p::yEGFP-SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0133 
                 pMeLS0079 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 249bp_CYC1p::yEGFP-SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0134 
                 pMeLS0080 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 209bp_CYC1p::yEGFP-SpHIS5 
               
               
                   
                 (Cen.PK113-5A) 
               
               
                 MeLS0135 
                 pMeLS0019 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 491bp_CYC1p_BenO_T1::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0136 
                 pMeLS0081 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 272bp_CYC1p_BenO_T1::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0137 
                 pMeLS0082 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 249bp_CYC1p_BenO_T1::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0138 
                 pMeLS0025 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 209bp_CYC1p_BenO_T1::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0177 
                 pMeLS0020 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 491bp_CYC1p_BenO_T2::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0259 
                 pMeLS0086 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 272bp_CYC1p_BenO_T2::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0260 
                 pMeLS0088 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 249bp_CYC1p_BenO_T2::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0180 
                 pMeLS0026 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 209bp_CYC1p_BenO_T2::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 MeLS0178 
                 pMeLS0021 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1/T2::yEGFP-SpHIS5 
               
               
                 MeLS0261 
                 pMeLS0087 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1/T2::yEGFP-SpHIS5 
               
               
                 MeLS0262 
                 pMeLS0089 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1/T2::yEGFP-SpHIS5 
               
               
                 MeLS0181 
                 pMeLS0027 + pCfB257 
                 mat a his3 leu2 trp1 KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1/T2::yEGFP-SpHIS5 
               
               
                 MeLS0164 
                 pMeLS0077 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0165 
                 pMeLS0078 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0166 
                 pMeLS0079 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0167 
                 pMeLS0080 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0156 
                 pMeLS0077 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0157 
                 pMeLS0078 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0158 
                 pMeLS0079 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0159 
                 pMeLS0080 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0160 
                 pMeLS0077 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0161 
                 pMeLS0078 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0162 
                 pMeLS0079 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0163 
                 pMeLS0080 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0139 
                 pMeLS0077 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0140 
                 pMeLS0078 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0141 
                 pMeLS0079 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0142 
                 pMeLS0080 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p::yEGFP-SpHIS5 
               
               
                 MeLS0172 
                 pMeLS0019 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0147 
                 pMeLS0081 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0173 
                 pMeLS0082 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0150 
                 pMeLS0025 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0168 
                 pMeLS0019 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0148 
                 pMeLS0081 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0169 
                 pMeLS0082 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0025 
                 pMeLS0025 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0170 
                 pMeLS0019 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0149 
                 pMeLS0081 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0171 
                 pMeLS0082 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0151 
                 pMeLS0025 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0143 
                 pMeLS0019 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0144 
                 pMeLS0081 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0145 
                 pMeLS0082 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0049 
                 pMeLS0025 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 MeLS0263 
                 pMeLS0020 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0190 
                 pMeLS0086 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0192 
                 pMeLS0088 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0264 
                 pMeLS0026 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0020 
                 pMeLS0020 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0182 
                 pMeLS0086 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0184 
                 pMeLS0088 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0026 
                 pMeLS0026 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0265 
                 pMeLS0020 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0194 
                 pMeLS0086 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0196 
                 pMeLS0088 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0266 
                 pMeLS0026 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0044 
                 pMeLS0020 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0186 
                 pMeLS0086 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0188 
                 pMeLS0088 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0050 
                 pMeLS0026 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T2::yEGFP-SpHIS5 
               
               
                 MeLS0267 
                 pMeLS0021 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0191 
                 pMeLS0087 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0193 
                 pMeLS0089 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0268 
                 pMeLS0027 + pMeLS0046 
                 mat a his3 leu2 trp1 TDH3p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0021 
                 pMeLS0021 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0183 
                 pMeLS0087 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0185 
                 pMeLS0089 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0027 
                 pMeLS0027 + pMeLS0044 
                 mat a his3 leu2 trp1 TEF1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0269 
                 pMeLS0021 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0195 
                 pMeLS0087 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0197 
                 pMeLS0089 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T/2::yEGFP-SpHIS5 
               
               
                 MeLS0270 
                 pMeLS0027 + pMeLS0053 
                 mat a his3 leu2 trp1 RNR2p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0045 
                 pMeLS0021 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 491bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0187 
                 pMeLS0087 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 272bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0189 
                 pMeLS0089 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 249bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0051 
                 pMeLS0027 + pMeLS0045 
                 mat a his3 leu2 trp1 REV1p::BenM-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_BenO_T1/2::yEGFP-SpHIS5 
               
               
                 MeLS0284 
                 pMeLS0025 + pMeLS0123 
                 mat a his3 leu2 trp1 REV1p::BenM(H110R, F211V, Y286N)- 
               
               
                   
                 (Cen.PK113-5A) 
                 KI.LEU2 209bp_CYC1p_BenO_T1::yEGFP-SpHIS5 
               
               
                 ST2377 
                 pCfB1237 + pCfB1239 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 (CCM 
                 (CEN.PK102-5B) 
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 
               
               
                 intermediate; 
               
               
                 with 
               
               
                 ScTkl1) 
               
               
                 ST3054 
                 pCfB1237 + pCfB2695 
                 mat a ura3 his3 leu2 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 (CCM 
                 (CEN.PK102-5B) 
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 
               
               
                 intermediate; 
               
               
                 no ScTkl1) 
               
               
                 ST3034 
                 pCfB1241 (ST2377) 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 
               
               
                 (CCM- 
                   
                 TDH3p::PaAroZ + TEF1p::CaCatA-KILEU2 + (TDH3p:: KpAroY.B 
               
               
                 multiple; 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n 
               
               
                 with ScTkl1) 
               
               
                 ST3059 
                 pCfB2696 (ST2377) 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 (CCM-single; 
                   
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 + TDH3p:: KpAroY.B 
               
               
                 with ScTkl1) 
                   
                 TEF1p::KpAroY.Ciso-KIURA3 
               
               
                 ST3058 
                 pCfB1241 (ST3054) 
                 mat a ura3 his3 leu2 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 (CCM- 
                   
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 + (TDH3p:: KpAroY.B 
               
               
                 multiple; no 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n 
               
               
                 ScTkl1) 
               
               
                 ST3154 
                 pCfB2696 (ST3054) 
                 mat a ura3 his3 leu2 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 (CCM-single; 
                   
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 + TDH3p:: KpAroY.B 
               
               
                 no ScTkl1) 
                   
                 TEF1p::KpAroY.Ciso-KIURA3 
               
               
                 ST4240- 
                 pCfB2553 + pCfB2764 
                 mat a URA3 HIS3 LEU2 TRP1 209bp_CYC1p_BenO_T1::yEGFP- 
               
               
                 1 (Reference 
                 (CEN.PK113-7D) 
                 HphMXsyn + REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 strain + 
               
               
                 biosensor) 
               
               
                 ST4241- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 1 (CCM- 
                 (ST3154) 
                 TDH3p::PaAroZ TEF1p::CaCatA KILEU2 + TDH3p:: KpAroY.B 
               
               
                 single; no 
                   
                 TEF1p::KpAroY.Ciso-KIURA3 + 209bp_CYC1p_BenO_T1::yEGFP 
               
               
                 ScTkl1 + 
                   
                 HphMXsyn + REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 biosensor) 
               
               
                 ST4242- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 8 (CCM- 
                 (ST3059) 
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 + TDH3p::KpAroY.B 
               
               
                 single; with 
                   
                 TEF1p::KpAroY.Ciso-KIURA3 + 209bp_CYC1p_BenO_T1::yEGFP 
               
               
                 ScTkl1 + 
                   
                 HphMXsyn + REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 biosensor) 
               
               
                 ST4243- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TEF1p::KpAroY.D-SpHIS5 + 
               
               
                 1 (CCM- 
                 (ST3058) 
                 TDH3p::PaAroZ TEF1p::CaCatA-KILEU2 + (TDH3p::KpAroY.B 
               
               
                 multiple; no 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n + 
               
               
                 ScTkl1 + 
                   
                 209bp_CYC1p_BenO_T1::yEGFP HphMXsyn + 
               
               
                 biosensor) 
                   
                 REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 ST4244- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 
               
               
                 1 (CCM- 
                 (ST3034) 
                 TDH3p::PaAroZ + TEF1p::CaCatA-KILEU2 + (TDH3p:: KpAroY.B 
               
               
                 multiple; 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n + 
               
               
                 with ScTkl1 + 
                   
                 209bp_CYC1p_BenO_T1::yEGFP HphMXsyn + 
               
               
                 biosensor) 
                   
                 REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 ST4244- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 
               
               
                 2 (CCM- 
                 (ST3034) 
                 TDH3p::PaAroZ + TEF1p::CaCatA-KILEU2 + (TDH3p:: KpAroY.B 
               
               
                 multiple; 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n + 
               
               
                 with ScTkl1 + 
                   
                 209bp_CYC1p_BenO_T1::yEGFP HphMXsyn + 
               
               
                 biosensor) 
                   
                 REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 ST4245- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 
               
               
                 2 (CCM- 
                 (ST3034) 
                 TDH3p::PaAroZ + TEF1p::CaCatA-KILEU2 + (TDH3p:: KpAroY.B 
               
               
                 multiple; 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n + 
               
               
                 with ScTkl1 + 
                   
                 209bp_CYC1p_BenO_T1::yEGFP HphMXsyn + 
               
               
                 biosensor) 
                   
                 REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 ST4246- 
                 pCfB2553 + pCfB2764 
                 mat a ura3 his3 leu2 TDH3p::ScTkl1 TEF1p::KpAroY.D-SpHIS5 
               
               
                 2 (CCM- 
                 (ST3034) 
                 TDH3p::PaAroZ + TEF1p::CaCatA-KILEU2 + (TDH3p:: KpAroY.B 
               
               
                 multiple; 
                   
                 TEF1p::KpAroY.Ciso-KIURA3tag) × n + 
               
               
                 with ScTkl1 + 
                   
                 209bp_CYC1p_BenO_T1::yEGFP HphMXsyn + 
               
               
                 biosensor) 
                   
                 REV1p::BenM(H110R, F211V, Y286N)-KanMXsyn 
               
               
                 TISNO-64 
                 pTS-27 + pTS-21 
                 mat a his3 leu2 trp1 REV1p::PcaQ-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_PcaO_T1::yEGFP-SpHIS5 
               
               
                 TISNO-66 
                 pTS-29 + pTS-23 
                 mat a his3 leu2 trp1 REV1p::ArgP-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_ArgO_T1::yEGFP-SpHIS5 
               
               
                 TISNO-67 
                 pTS-30 + pTS-24 + pTS-39 
                 mat a his3 leu2 trp1 REV1p::MdcR-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_MdcO_T1::yEGFP-SpHIS5 TEF1pr::SpMAE1 
               
               
                 TISNO-71 
                 pTS-33 + pTS-21 
                 mat a his3 leu2 trp1 TDH3p::PcaQ-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_PcaO_T1::yEGFP-SpHIS5 
               
               
                 TISNO-73 
                 pTS-35 + pTS-23 
                 mat a his3 leu2 trp1 TDH3p::ArgP-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_ArgO_T1::yEGFP-SpHIS5 
               
               
                 TISNO-74 
                 pTS-36 + pTS-24 + pTS-39 
                 mat a his3 leu2 trp1 TDH3p::MdcR-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_MdcO_T1::yEGFP-SpHIS5 TEF1pr::SpMAE1 
               
               
                 TISNO-79 
                 pCfB257 + pTS-21 
                 mat a his3 leu2 trp1 KI.LEU2 209bp_CYC1p_PcaO_T1::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 TISNO-81 
                 pCfB257 + pTS-23 
                 mat a his3 leu2 trp1 KI.LEU2 209bp_CYC1p_ArgO_T1::yEGFP- 
               
               
                   
                 (Cen.PK113-5A) 
                 SpHIS5 
               
               
                 TISNO-82 
                 pCfB257 + pTS-24 + pTS- 
                 mat a his3 leu2 trp1 KI.LEU2 209bp_CYC1p_MdcO_T1::yEGFP- 
               
               
                   
                 39 (Cen.PK113-5A) 
                 SpHIS5 TEF1pr::SpMAE1 
               
               
                 TISNO-83 
                 pCfB257 + pCfB2226 + 
                 mat a his3 leu2 trp1 KI.LEU2 Sp.HIS5syn 
               
               
                   
                 pTS-37 (Cen.PK113-5A) 
                 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn 
               
               
                 TISNO-89 
                 pTS-36 + pTS-23 
                 mat a his3 leu2 trp1 TDH3p::MdcR-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_ArgO_T1::yEGFP-SpHIS5 
               
               
                 TISNO-90 
                 pTS-35 + pTS-24 + pTS-39 
                 mat a his3 leu2 trp1 TDH3p::ArgP-KI.LEU2 
               
               
                   
                 (Cen.PK113-5A) 
                 209bp_CYC1p_MdcO_T1::yEGFP-SpHIS5 TEF1pr::SpMAE1 
               
               
                 TISNO-93 
                 pTS-45 + pTS-37 
                 mat a ura3 his3 leu2 TDH3p::FdeR-KI.URA3syn 
               
               
                   
                 (Cen.PK102-5B) 
                 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn 
               
               
                 TISNO-95 
                 pMije0124 + pTS-37 
                 mat a ura3 his3 leu2 REV1p::FdeR-KI.LEU2 
               
               
                   
                 (Cen.PK102-56) 
                 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn 
               
               
                 EV0 
                 — 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                   
                 aro10Δ0 
               
               
                 EV1 
                 pROP280 + pROP266 + 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                 pROP273 (EV0) 
                 aro10Δ0 + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS TEF1p::PhCHI PDC1p::At4Cl 
               
               
                 EV2 
                 pROP338 + pROP339 + 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                 pROP191 (EV1) 
                 aro10Δ0 + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS TEF1p::PhCHI PDC1p::At4Cl + TDH3p::AtPAL2 
               
               
                   
                   
                 PGK1p::HaCHS 
               
               
                 EV3 
                 pROP423 + pROP339 + 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                 pVAN968 (EV2) 
                 aro10Δ0 + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS TEF1p::PhCHI PDC1p::At4Cl + TDH3p::AtPAL2 
               
               
                   
                   
                 PGK1p::HaCHS + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS 
               
               
                 EVR0 (ctrl) 
                 pCfB2198 + pTS-49 (EV0) 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                   
                 aro10Δ0 + 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn1 + 
               
               
                   
                   
                 [CEN/ARS/URA3/TDH3p::FdeR] 
               
               
                 EVR1 
                 pCfB2198 + pTS-49 (EV1) 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                   
                 aro10Δ0 + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS TEF1p::PhCHI PDC1p::At4Cl + 
               
               
                   
                   
                 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn1 + 
               
               
                   
                   
                 [CEN/ARS/URA3/TDH3p::FdeR] 
               
               
                 EVR2 
                 pCfB2198 + pTS-49 (EV2) 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                   
                 aro10Δ0 + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS TEF1p::PhCHI PDC1p::At4Cl + TDH3p::AtPAL2 
               
               
                   
                   
                 PGK1p::HaCHS + 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn1 + 
               
               
                   
                   
                 [CEN/ARS/URA3/TDH3p::FdeR] 
               
               
                 EVR3 
                 pCfB2198 + pTS-49 (EV3) 
                 mat a ura3::LoxP-KanMX-LoxP pad1-fdc1::LoxP-NATMX-LoxP 
               
               
                   
                   
                 aro10Δ0 + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS TEF1p::PhCHI PDC1p::At4Cl + TDH3p::AtPAL2 
               
               
                   
                   
                 PGK1p::HaCHS + TDH3p::AtPAL2 TEF2p::C4H::L5::ATR2 
               
               
                   
                   
                 PGK1p::HaCHS + 209bp_CYC1p_FdeO_T1::yEGFP-hphMXsyn1 + 
               
               
                   
                   
                 [CEN/ARS/URA3/TDH3p::FdeR] 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Mean fluorescense intensities of GFP (24 h) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 CCM 
                 sd 
                 sd 
                 FC 
               
               
                 Strain ID 
                 Design 
                 Ctrl 
                 (1.4 mM) 
                 ctrl 
                 CCM 
                 (+/−CCM) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MeLS0081 
                 REV1p-BenM 
                 159 
                 175 
                 3 
                 7 
                 1.105042017 
               
               
                 MeLS0153 
                 RNR2p-BenM 
                 154 
                 174 
                 2 
                 2 
                 1.130151844 
               
               
                 MeLS0079 
                 TEF1p-BenM 
                 173 
                 185 
                 4 
                 12 
                 1.071428571 
               
               
                 MeLS0152 
                 TDH3p-BenM 
                 170 
                 184 
                 12 
                 3 
                 1.080392157 
               
               
                 MeLS0136 
                 272bp_CYC1p_BenO_T1:GFP 
                 155 
                 160 
                 0 
                 1 
                 1.032258065 
               
               
                 MeLS0162 
                 RNR2p-BenM + 249bp_CYC1p:GFP 
                 146 
                 164 
                 2 
                 4 
                 1.120728929 
               
               
                 MeLS0163 
                 RNR2p-BenM + 209bp_CYC1p:GFP 
                 146 
                 164 
                 4 
                 3 
                 1.120728929 
               
               
                 MeLS0138 
                 209bp_CYC1p_BenO_T1:GFP 
                 147 
                 167 
                 5 
                 4 
                 1.133484163 
               
               
                 MeLS0189 
                 REV1p-BenM + 
                 148 
                 181 
                 3 
                 8 
                 1.220224719 
               
               
                   
                 249bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0181 
                 209bp_CYC1p_BenO_T1/T2:GFP 
                 150 
                 165 
                 7 
                 6 
                 1.102222222 
               
               
                 MeLS0134 
                 209bp_CYC1p:GFP 
                 151 
                 171 
                 4 
                 4 
                 1.129955947 
               
               
                 MeLS0180 
                 209bp_CYC1p_BenO_T2:GFP 
                 152 
                 165 
                 3 
                 5 
                 1.087719298 
               
               
                 MeLS0159 
                 TEF1p-BenM + 209bp_CYC1p:GFP 
                 156 
                 177 
                 6 
                 7 
                 1.132478632 
               
               
                 MeLS0137 
                 249bp_CYC1p_BenO_T1:GFP 
                 156 
                 180 
                 1 
                 1 
                 1.151385928 
               
               
                 MeLS0050 
                 REV1p-BenM + 
                 156 
                 182 
                 6 
                 4 
                 1.166311301 
               
               
                   
                 209bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0158 
                 TEF1p-BenM + 249bp_CYC1p:GFP 
                 157 
                 175 
                 1 
                 2 
                 1.116772824 
               
               
                 MeLS0142 
                 REV1p-BenM + 209bp_CYC1p:GFP 
                 159 
                 174 
                 1 
                 2 
                 1.089958159 
               
               
                 MeLS0188 
                 REV1p-BenM + 
                 160 
                 194 
                 5 
                 5 
                 1.214583333 
               
               
                   
                 249bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0195 
                 RNR2p-BenM + 
                 161 
                 199 
                 6 
                 7 
                 1.238095238 
               
               
                   
                 272bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0197 
                 RNR2p-BenM + 
                 163 
                 256 
                 2 
                 15 
                 1.570552147 
               
               
                   
                 249bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0186 
                 REV1p-BenM + 
                 164 
                 197 
                 5 
                 3 
                 1.201219512 
               
               
                   
                 272bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0140 
                 REV1p-BenM + 272bp_CYC1p:GFP 
                 166 
                 183 
                 5 
                 3 
                 1.104627767 
               
               
                 MeLS0141 
                 REV1p-BenM + 249bp_CYC1p:GFP 
                 167 
                 176 
                 8 
                 3 
                 1.055888224 
               
               
                 MeLS0266 
                 RNR2p-BenM + 
                 167 
                 179 
                 5 
                 3 
                 1.071856287 
               
               
                   
                 209bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0161 
                 RNR2p-BenM + 272bp_CYC1p:GFP 
                 171 
                 180 
                 11 
                 8 
                 1.048638132 
               
               
                 MeLS0132 
                 272bp_CYC1p:GFP 
                 174 
                 211 
                 6 
                 7 
                 1.213051823 
               
               
                 MeLS0157 
                 TEF1p-BenM + 272bp_CYC1p:GFP 
                 175 
                 198 
                 8 
                 26 
                 1.12952381 
               
               
                 MeLS0167 
                 TDH3p-BenM + 209bp_CYC1p:GFP 
                 178 
                 194 
                 4 
                 12 
                 1.090056285 
               
               
                 MeLS0166 
                 TDH3p-BenM + 249bp_CYC1p:GFP 
                 181 
                 205 
                 1 
                 10 
                 1.130514706 
               
               
                 MeLS0182 
                 TEF1p-BenM + 
                 183 
                 211 
                 5 
                 11 
                 1.155109489 
               
               
                   
                 272bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0165 
                 TDH3p-BenM + 272bp_CYC1p:GFP 
                 187 
                 216 
                 2 
                 21 
                 1.156862745 
               
               
                 MeLS0171 
                 RNR2p-BenM + 
                 187 
                 290 
                 6 
                 4 
                 1.549019608 
               
               
                   
                 249bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0051 
                 REV1p-BenM + 
                 196 
                 307 
                 5 
                 3 
                 1.568994889 
               
               
                   
                 209bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0070 
                 RNR2p-BenM + 
                 200 
                 356 
                 3 
                 14 
                 1.775374376 
               
               
                   
                 209bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0190 
                 TDH3p-BenM + 
                 201 
                 218 
                 7 
                 7 
                 1.088039867 
               
               
                   
                 272bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0259 
                 272bp_CYC1p_BenO_T2:GFP 
                 201 
                 201 
                 4 
                 12 
                 1.001658375 
               
               
                 MeLS0262 
                 249bp_CYC1p_BenO_T1/T2:GFP 
                 203 
                 203 
                 7 
                 5 
                 1 
               
               
                 MeLS0187 
                 REV1p-BenM + 
                 207 
                 370 
                 7 
                 4 
                 1.789049919 
               
               
                   
                 272bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0145 
                 REV1p-BenM + 
                 207 
                 405 
                 2 
                 17 
                 1.953376206 
               
               
                   
                 249bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0133 
                 249bp_CYC1p:GFP 
                 210 
                 219 
                 11 
                 20 
                 1.042789223 
               
               
                 MeLS0196 
                 RNR2p-BenM + 
                 221 
                 349 
                 7 
                 10 
                 1.575301205 
               
               
                   
                 249bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0151 
                 RNR2p-BenM + 
                 230 
                 859 
                 6 
                 23 
                 3.734782609 
               
               
                   
                 209bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0049 
                 REV1p-BenM + 
                 231 
                 880 
                 37 
                 6 
                 3.80952381 
               
               
                   
                 209bp_CYC1p_BenO_T1:GFP 
               
               
                 MelS0194 
                 RNR2p-BenM + 
                 242 
                 258 
                 9 
                 13 
                 1.064649243 
               
               
                   
                 272bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0178 
                 491bp_CYC1p_BenO_T1/T2:GFP 
                 248 
                 241 
                 4 
                 3 
                 0.97311828 
               
               
                 MeLS0183 
                 TEF1p-BenM + 
                 259 
                 511 
                 4 
                 11 
                 1.969151671 
               
               
                   
                 272bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0184 
                 TEF1p-BenM + 
                 287 
                 475 
                 2 
                 8 
                 1.653132251 
               
               
                   
                 249bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0261 
                 272bp_CYC1p_BenO_T1/T2:GFP 
                 321 
                 257 
                 17 
                 2 
                 0.798755187 
               
               
                 MeLS0144 
                 REV1p-BenM + 
                 338 
                 804 
                 4 
                 23 
                 2.381046397 
               
               
                   
                 272bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0260 
                 249bp_CYC1p_BenO_T2:GFP 
                 344 
                 313 
                 11 
                 20 
                 0.909002904 
               
               
                 MeLS0264 
                 TDH3p-BenM + 
                 353 
                 527 
                 7 
                 5 
                 1.495274102 
               
               
                   
                 209bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0020 
                 TEF1p-BenM + 
                 365 
                 813 
                 14 
                 18 
                 2.224452555 
               
               
                   
                 491bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0191 
                 TDH3p-BenM + 
                 365 
                 691 
                 12 
                 43 
                 1.892335766 
               
               
                   
                 272bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0149 
                 RNR2p-BenM + 
                 369 
                 854 
                 21 
                 15 
                 2.313459801 
               
               
                   
                 272bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0026 
                 TEF1p-BenM + 
                 380 
                 406 
                 5 
                 8 
                 1.068481124 
               
               
                   
                 209bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0193 
                 TDH3p-BenM + 
                 407 
                 1630 
                 10 
                 67 
                 4.004095004 
               
               
                   
                 249bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0044 
                 REV1p-BenM + 
                 438 
                 667 
                 18 
                 7 
                 1.523990861 
               
               
                   
                 491bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0265 
                 RNR2p-BenM + 
                 461 
                 698 
                 22 
                 21 
                 1.51300578 
               
               
                   
                 491bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0027 
                 TEF1p-BenM + 
                 554 
                 1879 
                 60 
                 20 
                 3.393738712 
               
               
                   
                 209bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0192 
                 TDH3p-BenM + 
                 555 
                 978 
                 13 
                 22 
                 1.760504202 
               
               
                   
                 249bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0263 
                 TDH3p-BenM + 
                 631 
                 1197 
                 15 
                 15 
                 1.897517169 
               
               
                   
                 491bp_CYC1p_BenO_T2:GFP 
               
               
                 MeLS0177 
                 491bp_CYC1p_BenO_T2:GFP 
                 641 
                 698 
                 18 
                 7 
                 1.088403536 
               
               
                 MeLS0268 
                 TDH3p-BenM + 
                 668 
                 2172 
                 14 
                 96 
                 3.250374065 
               
               
                   
                 209bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0169 
                 TEF1p-BenM + 
                 672 
                 2913 
                 12 
                 21 
                 4.337468983 
               
               
                   
                 249bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0135 
                 491bp_CYC1p_BenO_T1:GFP 
                 754 
                 801 
                 12 
                 11 
                 1.061864781 
               
               
                 MeLS0173 
                 TDH3p-BenM + 
                 755 
                 3554 
                 6 
                 78 
                 4.706843267 
               
               
                   
                 249bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0267 
                 TDH3p-BenM + 
                 840 
                 2218 
                 33 
                 81 
                 2.639825466 
               
               
                   
                 491bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0148 
                 TEF1p-BenM + 
                 1164 
                 4032 
                 39 
                 145 
                 3.463212139 
               
               
                   
                 272bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0025 
                 TEF1p-BenM + 
                 1185 
                 4139 
                 6 
                 85 
                 3.493528419 
               
               
                   
                 209bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0150 
                 TDH3p-BenM + 
                 1192 
                 4868 
                 16 
                 47 
                 4.082471345 
               
               
                   
                 209bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0045 
                 REV1p-BenM + 
                 1197 
                 1698 
                 8 
                 67 
                 1.418151448 
               
               
                   
                 491bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0269 
                 RNR2p-BenM + 
                 1291 
                 2589 
                 80 
                 74 
                 2.005422153 
               
               
                   
                 491bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0147 
                 TDH3p-BenM + 
                 1333 
                 4984 
                 28 
                 65 
                 3.738 
               
               
                   
                 272bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0021 
                 TEF1p-BenM + 
                 1358 
                 2967 
                 16 
                 142 
                 2.18404908 
               
               
                   
                 491bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0139 
                 REV1p-BenM + 491bp_CYC1p:GFP 
                 2270 
                 2231 
                 1813 
                 1771 
                 0.98281686 
               
               
                 MeLS0160 
                 RNR2p-BenM + 491bp_2YC1p:GFP 
                 2625 
                 2811 
                 39 
                 118 
                 1.07059421 
               
               
                 MeLS0185 
                 TEF1p-BenM + 
                 2632 
                 9992 
                 273 
                 294 
                 3.796833439 
               
               
                   
                 249bp_CYC1p_BenO_T1/T2:GFP 
               
               
                 MeLS0131 
                 491bp_CYC1p:GFP 
                 2781 
                 2899 
                 30 
                 116 
                 1.042435867 
               
               
                 MeLS0156 
                 TEF1p-BenM + 491bp_CYC1p:GFP 
                 3046 
                 3016 
                 44 
                 67 
                 0.990152095 
               
               
                 MeLS0164 
                 TDH3p-BenM + 491bp_CYC1p:GFP 
                 4003 
                 4509 
                 90 
                 105 
                 1.126488467 
               
               
                 MeLS0143 
                 REV1p-BenM + 
                 5321 
                 5789 
                 181 
                 229 
                 1.0880842 
               
               
                   
                 491bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0170 
                 RNR2p-BenM + 
                 5616 
                 6114 
                 87 
                 48 
                 1.088734568 
               
               
                   
                 491bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0172 
                 TDH3p-BenM + 
                 6894 
                 10605 
                 24 
                 353 
                 1.53834252 
               
               
                   
                 491bp_CYC1p_BenO_T1:GFP 
               
               
                 MeLS0168 
                 TEF1p-BenM + 
                 15429 
                 20250 
                 355 
                 316 
                 1.312441938 
               
               
                   
                 491bp_CYC1p_BenO_T1:GFP 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 List of plasmids 
               
            
           
           
               
               
               
               
            
               
                 Plasmid 
                 Parent 
                   
                   
               
               
                 name 
                 plasmid 
                 Description 
                 Reference/Source 
               
               
                   
               
               
                 pCfB258 
                 — 
                 pX-4-LoxP-SpHIS5 
                 Jensen et al., 2014 
               
               
                 pCfB322 
                 — 
                 pTY4-LoxP-KIURA3tag 
                 Borodina et al., 2014 
               
               
                 pCfB388 
                 — 
                 pXI-1-LoxP-KILEU2 
                 Jensen et al., 2014 
               
               
                 pCfB390 
                 — 
                 pXI-3-LoxP-KIURA3 
                 Jensen et al., 2014 
               
               
                 pCfB2198 
                 — 
                 pXII-4-LoxP-HphMXsyn 
                 Stovicek et al., 2015 
               
               
                 pCfB2223 
                 — 
                 pX-3-LoxP-KanMXsyn 
                 Stovicek et al., 2015 
               
               
                 pCfB2226 
                 — 
                 pX-4-IoxP-SpHIS5syn 
                 Stovicek et al., 2015 
               
               
                 pCf61237 
                 pCfB258 
                 pX-4-IoxP-SpHiS5-ScTkl1&lt;-TDH3p-TEF1p-&gt; 
                 This study 
               
               
                   
                   
                 KpAroY.D 
               
               
                 pCfB1239 
                 pCfB388 
                 pXI-1-LoxP-KILEU2-PaAroZ&lt;-TDH3p-TEF1p-&gt; 
                 This study 
               
               
                   
                   
                 CaCatA 
               
               
                 pCfB1241 
                 pCfB322 
                 pTY4-LoxP-KIURA3tag-KpAroY.B&lt;-TDH3p- 
                 This study 
               
               
                   
                   
                 TEF1p-&gt;KpAroY.Ciso 
               
               
                 pCfB2374 
                 — 
                 pXI-1-IoxP-KIURA3syn 
                 Stovicek et al., 2015 
               
               
                 pCfB3039 
                 — 
                 pXII-2 
                 Stovicek et al., 2015 
               
               
                 pCfB2695 
                 pCfB258 
                 pX-4-LoxP-SpHiS5-TEF1p-&gt;KpAroY.D 
                 This study 
               
               
                 pCfB2696 
                 pCfB390 
                 pXI-3-KIURA3-KpAroY.6&lt;-TDH3p-TEF1p-&gt; 
                 This study 
               
               
                   
                   
                 KpAroY.Ciso 
               
               
                 pCfB2553 
                 pCfB2198 
                 pXII-4-LoxP-HphMXsyn- 
                 This study 
               
               
                   
                   
                 209bp_CYC1p_BenO_T1-&gt;yEGFP 
               
               
                 pCfB2764 
                 pCfB2223 
                 pX-3-LoxP-KanMXsyn-REV1p-&gt;BenM(H110R, 
                 This study 
               
               
                   
                   
                 F211V, Y286N) 
               
               
                 pCfB257 
                 — 
                 pX-3-LoxP-KILEU2 
                 Jensen et al., 2014 
               
               
                 pCfB262 
                 — 
                 pXII-4-LoxP-SpHIS5 
                 Jensen et al., 2014 
               
               
                 pRS416U 
                 — 
                 URA3, USER cassette 
                 This study 
               
               
                 pMeLS0045 
                 pCfB257 
                 pX-3-LoxP-KILEU2-REV1p-&gt;BenM 
                 This study 
               
               
                 pMeLS0044 
                 pCfB257 
                 pX-3-LoxP-KILEU2-TEF1p-&gt;BenM 
                 This study 
               
               
                 pMeLS0053 
                 pCfB257 
                 pX-3-LoxP-KILEU2-RNR2p-&gt;BenM 
                 This study 
               
               
                 pMeLS0046 
                 pCfB257 
                 pX-3-LoxP-KILEU2-TDH3p-&gt;BenM 
                 This study 
               
               
                 pMeLS0077 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-491bp_CYC1p-&gt;yeGFP 
                 This study 
               
               
                 pMeLS0078 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-272bp_CYC1p-&gt;yeGFP 
                 This study 
               
               
                 pMeLS0079 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-249bp_CYC1p-&gt;yeGFP 
                 This study 
               
               
                 pMeLS0080 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-209bp_CYC1p-&gt;yeGFP 
                 This study 
               
               
                 pMeLS0019 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-491bp_CYC1p_BenO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0081 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-272bp_CYC1p_BenO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0082 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-249bp_CYC1p_BenO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0025 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-209bp_CYC1p_BenO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0020 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-491bp_CYC1p_BenO_T2-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0086 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-272bp_CYC1p_BenO_T2-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0088 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-249bp_CYC1p_BenO_T2-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0026 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-209bp_CYC1p_BenO_T2-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pMeLS0021 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5- 
                 This study 
               
               
                   
                   
                 491bp_CYC1p_BenO_T1/2-&gt;yeGFP 
               
               
                 pMeLS0087 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5- 
                 This study 
               
               
                   
                   
                 272bp_CYC1p_BenO_T1/2-&gt;yeGFP 
               
               
                 pMeLS0089 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5- 
                 This study 
               
               
                   
                   
                 249bp_CYC1p_BenO_T1/2-&gt;yeGFP 
               
               
                 pMeLS0027 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5- 
                 This study 
               
               
                   
                   
                 209bp_CYC1p_BenO_T1/2-&gt;yeGFP 
               
               
                 pMeLS0123 
                 pCfB257 
                 pX-3-LoxP-KILEU2-REV1p-&gt;BenM(H110R, 
                 This study 
               
               
                   
                   
                 F211V, Y286N) 
               
               
                 pMije0124 
                 pCfB257 
                 pX-3-LoxP-KILEU2-REV1p-&gt;FdeR 
                 This study 
               
               
                 pTS-21 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-209bp_CYC1p_PcaO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pTS-23 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-209bp_CYC1p_ArgO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pTS-24 
                 pCfB262 
                 pXII-4-LoxP-SpHIS5-209bp_CYC1p_MdcO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pTS-27 
                 pCfB257 
                 pX-3-LoxP-KILEU2-REV1p-&gt;PcaQ 
                 This study 
               
               
                 pTS-29 
                 pCfB257 
                 pX-3-LoxP-KILEU2-REV1p-&gt;ArgP 
                 This study 
               
               
                 pTS-30 
                 pCfB257 
                 pX-3-LoxP-KILEU2-REV1p-&gt;MdcR 
                 This study 
               
               
                 pTS-33 
                 pCfB257 
                 pX-3-LoxP-KILEU2-TDH3p-&gt;PcaQ 
                 This study 
               
               
                 pTS-35 
                 pCfB257 
                 pX-3-LoxP-KILEU2-TDH3p-&gt;ArgP 
                 This study 
               
               
                 pTS-36 
                 pCfB257 
                 pX-3-LoxP-KILEU2-TDH3p-&gt;MdcR 
                 This study 
               
               
                 pTS-37 
                 pCfB2198 
                 pXII-4-LoxP-HphMX-209bp_CYC1p_FdeO_T1-&gt; 
                 This study 
               
               
                   
                   
                 yeGFP 
               
               
                 pTS-38 
                 pCfB2374 
                 pXI-1-LoxP-KI.URA3syn-TDH3p-&gt;FdeR 
                 This study 
               
               
                 pTS-39 
                 pCfB3039 
                 pXII-2-TEF1p-&gt;Sp.MAE1 
                 This study 
               
               
                 pTS-49 
                 pRS416U 
                 pURA3-TDH3p-&gt;FdeR 
                 This study 
               
               
                 pROP280 
                 — 
                 pX-DR-KILEU2-AtPAL2&lt;-TDH3p-TEF2p-&gt; 
                 This study 
               
               
                   
                   
                 AtC4H::L5::AtATR2 
               
               
                 pROP266 
                 — 
                 HaCHS&lt;-PGK1p-TEF1p-&gt;PhCHI 
                 This study 
               
               
                 pROP273 
                 — 
                 pX-PDC1p-&gt;At4CL2 
                 This study 
               
               
                 pROP338 
                 — 
                 pXI-DR-KILEU2-AtPAL2&lt;-TDH3p 
                 This study 
               
               
                 pROP339 
                 — 
                 HaCHS&lt;-PGK1p 
                 This study 
               
               
                 pROP191 
                 — 
                 pXI 
                 This study 
               
               
                 pROP423 
                 — 
                 pXVI-DR-KILEU2-AtPAL2&lt;-TDH3p-TEF2p-&gt; 
                 This study 
               
               
                   
                   
                 AtC4H::L5::AtATR2 
               
               
                 pVAN968 
                 — 
                 pXVI 
                 This study 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 List of synthetic genes. For synthetic reporter promoters based on the 
               
               
                 209 bp_CYC1p scaffold promoter, LTTR operator sites are marked in bold and start 
               
               
                 codon for yeGFP is marked in green. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 LOCUS CaCatA 912 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGTCCCAAG CTTTCACCGA ATCTGTTAAG ACTTCTTTGG GTCCAAATGC TACTCCAAGA 
               
               
                   
               
               
                  61 
                 GCTAAAAAGT TGATTGCCTC TTTGGTTCAA CACGTTCATG ATTTCGCTAG AGAAAACCAT 
               
               
                   
               
               
                 121 
                 TTGACTACCG AAGATTGGTT GTGGGGTGTT GATTTCATTA ACAGAATTGG TCAAATGTCC 
               
               
                   
               
               
                 181 
                 GACTCCAGAA GAAACGAAGG TATTTTGGTT TGCGATATCA TCGGTTTGGA AACCTTGGTT 
               
               
                   
               
               
                 241 
                 GATGCTTTGA CTAACGAATC CGAACAATCT AACCATACCT CCTCTGCTAT TTTGGGTCCT 
               
               
                   
               
               
                 301 
                 TTTTACTTGC CAGATTCTCC AGTTTATCCA AACGGTGGTT CTATCGTTCA AAAGGCTATT 
               
               
                   
               
               
                 361 
                 CCAACTGATG TTAAGTGCTT CGTTAGAGGT AAGGTTACTG ATACTGAAGG TAAACCATTG 
               
               
                   
               
               
                 421 
                 GGTGGTGCTC AATTGGAAGT TTGGCAATGT AATTCTGCTG GTTTCTACTC TCAACAAGCT 
               
               
                   
               
               
                 481 
                 GATCATGATG GTCCAGAATT CAATTTGAGA GGTACTTTCA TTACCGACGA CGAAGGTAAT 
               
               
                   
               
               
                 541 
                 TACTCCTTCG AATGTTTAAG ACCAACCTCC TATCCAATTC CATACGATGG TCCTGCTGGT 
               
               
                   
               
               
                 601 
                 GATTTGTTGA AAATCATGGA TAGACATCCA AACAGACCAT CCCATATTCA TTGGAGAGTT 
               
               
                   
               
               
                 661 
                 TCTCATCCAG GTTACCATAC TTTGATCACC CAAATCTATG ATGCTGAATG TCCATACACC 
               
               
                   
               
               
                 721 
                 AACAACGATT CTGTTTACGC TGTTAAGGAT GACATCATCG TTCACTTCGA AAAGGTTGAT 
               
               
                   
               
               
                 781 
                 AACAAGGATA AGGATTTGGT CGGTAAGGTC GAATACAAGT TGGATTACGA TATTTCCTTG 
               
               
                   
               
               
                 841 
                 GCCACCGAAT CCTCTATTCA AGAAGCTAGA GCTGCTGCTA AAGCTAGACA AGATGCTGAA 
               
               
                   
               
               
                 901 
                 ATCAAGTTGT AA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS KpAroY.B 594 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGAAGTTGA TCATCGGTAT GACTGGTGCT ACAGGTGCTC CATTGGGTGT TGCTTTGTTG 
               
               
                   
               
               
                  61 
                 CAAGCTTTGA GAGATATGCC AGAAGTTGAA ACCCATTTGG TTATGTCTAA ATGGGCTAAG 
               
               
                   
               
               
                 121 
                 ACCACCATTG AATTGGAAAC TCCATGGACT GCTAGAGAAG TTGCTGCTTT GGCTGATTTT 
               
               
                   
               
               
                 181 
                 TCTCATTCTC CAGCTGATCA AGCTGCTACT ATTTCTTCTG GTTCTTTCAG AACTGATGGT 
               
               
                   
               
               
                 241 
                 ATGATCGTTA TTCCATGCTC TATGAAAACC TTGGCTGGTA TTAGAGCTGG TTATGCTGAA 
               
               
                   
               
               
                 301 
                 GGTTTGGTTG GTAGAGCTGC TGATGTTGTT TTGAAAGAAG GTAGAAAGTT GGTCTTGGTC 
               
               
                   
               
               
                 361 
                 CCAAGAGAAA TGCCATTGTC TACTATCCAT TTGGAAAACA TGTTGGCCTT GTCTAGAATG 
               
               
                   
               
               
                 421 
                 GGTGTAGCTA TGGTTCCACC AATGCCAGCT TATTACAATC ATCCAGAAAC CGTTGATGAC 
               
               
                   
               
               
                 481 
                 ATCACCAACC ATATAGTTAC CAGAGTTTTG GACCAATTCG GTTTGGATTA TCACAAAGCT 
               
               
                   
               
               
                 541 
                 AGAAGATGGA ACGGTTTGAG AACTGCTGAA CAATTCGCTC AAGAAATTGA ATGA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS KpAroY.Ciso 1509 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGACCGCCC CAATCCAAGA TTTGAGAGAT GCTATTGCTT TGTTACAACA ACACGACAAT 
               
               
                   
               
               
                   61 
                 CAATACTTGG AAACCGATCA TCCAGTTGAT CCAAATGCTG AATTGGCTGG TGTTTACAGA 
               
               
                   
               
               
                  121 
                 CATATTGGTG CTGGTGGTAC TGTAAAAAGA CCAACTAGAA TTGGTCCAGC CATGATGTTC 
               
               
                   
               
               
                  181 
                 AACAACATTA AGGGTTATCC ACACTCCAGA ATCTTGGTTG GTATGCATGC TTCTAGACAA 
               
               
                   
               
               
                  241 
                 AGAGCAGCTT TGTTGTTGGG TTGTGAAGCT TCTCAATTGG CTTTGGAAGT TGGTAAAGCT 
               
               
                   
               
               
                  301 
                 GTTAAGAAAC CAGTTGCTCC AGTTGTTGTT CCAGCTTCTT CTGCTCCATG TCAAGAACAA 
               
               
                   
               
               
                  361 
                 ATTTTCTTGG CTGATGATCC AGACTTCGAT TTGAGAACTT TGTTGCCAGC TCATACCAAC 
               
               
                   
               
               
                  421 
                 ACTCCAATTG ATGCTGGTCC ATTTTTTTGT TTGGGTTTGG CTTTAGCTTC TGATCCTGTT 
               
               
                   
               
               
                  481 
                 GATGCTTCTT TGACCGATGT TACCATTCAT AGATTGTGCG TTCAAGGTAG AGATGAATTG 
               
               
                   
               
               
                  541 
                 TCTATGTTTT TGGCTGCCGG TAGACATATC GAAGTTTTTA GACAAAAAGC TGAAGCTGCT 
               
               
                   
               
               
                  601 
                 GGTAAGCCAT TGCCAATTAC TATTAACATG GGTTTAGATC CAGCCATCTA CATTGGTGCT 
               
               
                   
               
               
                  661 
                 TGTTTTGAAG CTCCAACTAC TCCATTTGGT TACAACGAAT TGGGTGTTGC TGGTGCTTTG 
               
               
                   
               
               
                  721 
                 AGACAAAGAC CAGTTGAATT GGTTCAAGGT GTTTCTGTTC CAGAAAAGGC TATTGCTAGA 
               
               
                   
               
               
                  781 
                 GCCGAAATAG TTATCGAAGG TGAATTATTG CCAGGTGTCA GAGTTAGAGA AGATCAACAT 
               
               
                   
               
               
                  841 
                 ACAAATTCCG GTCATGCTAT GCCAGAATTT CCAGGTTATT GTGGTGGTGC TAATCCATCT 
               
               
                   
               
               
                  901 
                 TTGCCAGTTA TTAAGGTTAA GGCCGTTACC ATGAGAAACA ACGCTATTTT ACAAACTTTG 
               
               
                   
               
               
                  961 
                 GTCGGTCCAG GTGAAGAACA TACAACTTTG GCTGGTTTGC CAACCGAAGC TTCTATTTGG 
               
               
                   
               
               
                 1021 
                 AATGCTGTTG AAGCTGCAAT TCCAGGTTTC TTGCAAAATG TTTATGCTCA TACAGCTGGT 
               
               
                   
               
               
                 1081 
                 GGTGGTAAGT TCTTGGGTAT ATTGCAAGTC AAGAAAAGAC AACCAGCTGA CGAAGGTAGA 
               
               
                   
               
               
                 1141 
                 CAAGGTCAAG CTGCTTTATT AGCTTTGGCT ACTTACTCCG AATTGAAGAA TATCATCTTG 
               
               
                   
               
               
                 1201 
                 GTCGATGAAG ATGTTGATAT CTTCGATTCC GATGATATTT TGTGGGCTAT GACTACTAGA 
               
               
                   
               
               
                 1261 
                 ATGCAAGGTG ATGTTTCCAT TACTACCATT CCAGGTATTA GAGGTCACCA ATTAGATCCA 
               
               
                   
               
               
                 1321 
                 TCTCAAACCC CAGAATACTC CCCATCAATT AGAGGTAATG GTATCTCCTG TAAGACCATT 
               
               
                   
               
               
                 1381 
                 TTCGATTGCA CTGTTCCATG GGCTTTGAAG TCTCATTTTG AAAGAGCACC ATTTGCTGAC 
               
               
                   
               
               
                 1441 
                 GTTGATCCTA GACCTTTTGC TCCAGAATAT TTCGCTAGAT TGGAAAAGAA TCAAGGTTCC 
               
               
                   
               
               
                 1501 
                 GCTAAGTAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS KpAroY.D 237 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGATCTGTC CAAGATGCGC CGACGAAAAA ATTGAAGTTA TGGCTACTTC TCCAGTTAAG 
               
               
                   
               
               
                  61 
                 GGTGTTTGGA CTGTTTATCA ATGTCAACAC TGCTTGTACA CTTGGAGAGA TACTGAACCA 
               
               
                   
               
               
                 121 
                 TTGAGAAGAA CCTCTAGAGA ACATTACCCT GAAGCTTTCA GAATGACCCA AAAGGATATT 
               
               
                   
               
               
                 181 
                 GATGAAGCTC CACAAGTTCC TCATGTTCCA CCATTATTGC CAGAAGATAA GAGATAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS PaAroZ 1104 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGCCATCCA AGTTGGCCAT TACCTCTATG TCTTTGGGTA GATGTTATGC CGGTCATTCT 
               
               
                   
               
               
                   61 
                 TTCACTACTA AGTTGGATAT GGCTAGAAAG TACGGTTACC AAGGTTTGGA ATTATTCCAT 
               
               
                   
               
               
                  121 
                 GAAGATTTGG CTGATGTCGC CTATAGATTG TCTGGTGAAA CTCCATCTCC ATGTGGTCCA 
               
               
                   
               
               
                  181 
                 TCACCAGCTG CTCAATTGTC TGCTGCTAGA CAAATTTTGA GAATGTGCCA AGTCAGAAAC 
               
               
                   
               
               
                  241 
                 ATCGAAATCG TTTGCTTGCA ACCATTCTCT CAATACGATG GTTTGTTGGA TAGAGAAGAA 
               
               
                   
               
               
                  301 
                 CACGAAAGAA GATTGGAACA ATTGGAATTC TGGATCGAAT TGGCCCATGA ATTGGATACC 
               
               
                   
               
               
                  361 
                 GATATTATTC AAATTCCAGC CAACTTCTTG CCAGCCGAAG AAGTTACTGA AGATATCTCT 
               
               
                   
               
               
                  421 
                 TTGATTGTCT CCGACTTGCA AGAAGTAGCT GATATGGGTT TACAAGCTAA CCCACCAATT 
               
               
                   
               
               
                  481 
                 AGATTCGTTT ACGAAGCTTT GTGTTGGTCC ACTAGAGTTG ATACTTGGGA AAGATCTTGG 
               
               
                   
               
               
                  541 
                 GAAGTTGTTC AAAGAGTTAA CAGACCAAAC TTCGGTGTTT GTTTGGACAC TTTTAACATT 
               
               
                   
               
               
                  601 
                 GCCGGTAGAG TTTATGCTGA TCCAACTGTT GCTTCTGGTA GAACTCCAAA TGCTGAAGAA 
               
               
                   
               
               
                  661 
                 GCTATCAGAA AGTCCATTGC CAGATTGGTT GAAAGAGTTG ACGTTTCCAA GGTTTTCTAC 
               
               
                   
               
               
                  721 
                 GTTCAAGTTG TTGATGCCGA AAAGTTGAAG AAACCATTGG TTCCAGGTCA CAGATTCTAT 
               
               
                   
               
               
                  781 
                 GATCCAGAAC AACCAGCTAG AATGTCTTGG TCTAGAAACT GCAGATTATT CTACGGTGAA 
               
               
                   
               
               
                  841 
                 AAGGATAGAG GTGCTTACTT GCCAGTAAAA GAAATTGCTT GGGCTTTTTT CAACGGTTTG 
               
               
                   
               
               
                  901 
                 GGTTTTGAAG GTTGGGTTTC CTTAGAATTA TTCAACAGAA GAATGTCCGA TACCGGTTTT 
               
               
                   
               
               
                  961 
                 GGTGTTCCAG AAGAATTAGC TAGAAGAGGT GCTGTTTCTT GGGCTAAATT GGTTAGAGAT 
               
               
                   
               
               
                 1021 
                 ATGAAGATCA CCGTTGACTC TCCAACTCAA CAACAAGCTA CACAACAACC TATCAGAATG 
               
               
                   
               
               
                 1081 
                 TTGTCTTTGT CAGCTGCTTT GTGA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS BenM 915 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGGAATTGA GACACTTGAG ATACTTCGTT GCCGTTGTTG AAGAACAATC TTTTACAAAG 
               
               
                   
               
               
                  61 
                 GCTGCCGACA AGTTGTGTAT TGCTCAACCA CCATTATCCA GACAAATCCA AAACTTGGAA 
               
               
                   
               
               
                 121 
                 GAAGAATTGG GTATCCAATT ATTGGAAAGA GGTTCCAGAC CAGTTAAGAC TACTCCAGAA 
               
               
                   
               
               
                 181 
                 GGTCATTTCT TTTACCAATA CGCCATCAAG TTGTTGTCCA ACGTTGATCA AATGGTCAGT 
               
               
                   
               
               
                 241 
                 ATGACCAAGA GAATTGCCTC TGTTGAAAAG ACCATTAGAA TCGGTTTTGT TGGTTCCTTG 
               
               
                   
               
               
                 301 
                 TTGTTCGGTT TGTTGCCAAG AATTATCCAC TTGTACAGAC AAGCTCATCC AAACTTGAGA 
               
               
                   
               
               
                 361 
                 ATCGAATTAT ACGAAATGGG TACTAAGGCT CAAACCGAAG CTTTGAAAGA AGGTAGAATT 
               
               
                   
               
               
                 421 
                 GACGCTGGTT TTGGTAGATT GAAGATTTCT GATCCAGCCA TCAAGAGAAC CTTGTTGAGA 
               
               
                   
               
               
                 481 
                 AACGAAAGAT TGATGGTTGC TGTTCATGCT TCCCATCCAT TGAATCAAAT GAAGGATAAG 
               
               
                   
               
               
                 541 
                 GGTGTTCACT TGAACGATTT GATCGACGAA AAGATCTTGT TGTACCCATC TTCTCCAAAG 
               
               
                   
               
               
                 601 
                 CCAAACTTCT CTACTCATGT TATGAACATC TTCTCTGACC ATGGTTTGGA ACCTACCAAG 
               
               
                   
               
               
                 661 
                 ATTAACGAAG TTAGAGAAGT CCAATTGGCC TTGGGTTTGG TTGCTGCTGG TGAAGGTATT 
               
               
                   
               
               
                 721 
                 TCATTGGTTC CAGCTTCTAC CCAATCCATT CAATTATTCA ACTTGTCCTA CGTCCCATTA 
               
               
                   
               
               
                 781 
                 TTAGATCCAG ATGCTATTAC CCCAATCTAC ATTGCTGTTA GAAACATGGA AGAATCCACC 
               
               
                   
               
               
                 841 
                 TACATCTACT CATTATACGA AACCATCAGA CAAATCTACG CCTACGAAGG TTTTACTGAA 
               
               
                   
               
               
                 901 
                 CCACCAAATT GGTAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS FdeR 930 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGCGTTTCA ACAAGCTCGA CCTCAATCTT CTGGTCGCCC TGGATGCACT GCTCACGGAG 
               
               
                   
               
               
                  61 
                 ATGAGCATCA GCCGCGCCGC CGAAAAGATC CATCTGAGCC AGTCGGCCAT GAGCAATGCC 
               
               
                   
               
               
                 121 
                 CTGGCGCGGC TGCGCGAGTA TTTCGATGAT GAATTGCTGA TCCAGGTGGG CCGGCGCATG 
               
               
                   
               
               
                 181 
                 GAGCCCACGC CGCGCGCCGA GGTGCTCAAG GATGCGGTGC ATGATGTGCT GCGGCGTATC 
               
               
                   
               
               
                 241 
                 GATGGCTCCA TCGCGGCGCT GCCGGCCTTC GTGCCGGCCG AGTCCACGCG CGAGTTTCGC 
               
               
                   
               
               
                 301 
                 ATCTCGGTTT CGGACTTTAC GCTCTCCGTC CTCATCCCCC GGGTGCTGGC GCGCGCGCAC 
               
               
                   
               
               
                 361 
                 GCCGAGGGCA AGCACATCCG CTTTGCCCTG ATGCCGCAGG TGCAAGACCC GACCCGCTCG 
               
               
                   
               
               
                 421 
                 CTGGATCGGG CCGAGGTGGA CCTGCTGGTC TTGCCGCAGG AATTCTGCAC GCCCGATCAT 
               
               
                   
               
               
                 481 
                 CCTGCCGAAG AGGTCTTCCG CGAACGGCAT GTCTGCGTGG TCTGGCGCGA CAGTGCGCTG 
               
               
                   
               
               
                 541 
                 GCGCAAGGCG AGCTGACGCT GGAACGCTAC ATGGCCTCAG GCCATGTGGT GATGGTGCCG 
               
               
                   
               
               
                 601 
                 CCTGGGGCCA ATGCGTCGTC GGTGGAGGCG TGGATGGCCA GGAAGCTGGG CTTTGCGCGC 
               
               
                   
               
               
                 661 
                 CGGGTGGAAG TGACCAGCTT CAGCTTCGCT TCTGCGCTGG CGCTGGTACA GGGGACGGAC 
               
               
                   
               
               
                 721 
                 CGCATCGCCA CGGTGCATGC CCGGCTGGCG CAGCTGCTGG CTCCGCAATG GCCGGTGGTG 
               
               
                   
               
               
                 781 
                 ATCAAGGAGA GTCCGCTGTC GCTGGGCGAG ATGCGGCAGA TGATGCAGTG GCATCGCTAC 
               
               
                   
               
               
                 841 
                 CGCAGCAATG ATCCTGGCAT CCAGTGGCTG CGTCGGGTGT TTCTGGAGAG TGCGCAGGAG 
               
               
                   
               
               
                 901 
                 ATGGATGCGG CGCTGCCAGG CATCTGCTGA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS PcaQ 942 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGATTGATG CACGTGTGAA ATTTAGACAT TTGCAAACTT TTGTAGAAGT TGCTAGACAA 
               
               
                   
               
               
                  61 
                 AAGAGTGTTG TAAAAGCAGC CGAATTATTA CATGTAACAC AGCCAGCAGT GACTAAGACC 
               
               
                   
               
               
                 121 
                 ATAAGGGAAT TGGAAGAGGT ATTAGGTGTC GCCGTGTTTG AAAGAGAAGG TCGTGGTATC 
               
               
                   
               
               
                 181 
                 AAAATAACAA GATATGGGGA AGTTTTTTTG AGACATGCAG GAGCTGCCCT TACGGCTCTT 
               
               
                   
               
               
                 241 
                 CGTCAAGGTC TAGACAGCGT ATCTCAAGAA AGAAGTGGCG AAGGTCCACC AATCAGGGTA 
               
               
                   
               
               
                 301 
                 GGCGCCTTAC CTACAGTATC AACTAGAATC ATGCCAAGAG CTATTGCACT TTTTCTGAAG 
               
               
                   
               
               
                 361 
                 GAAAAAACGG GTGCAAGAAT TAAAATAGTC ACAGGCGAAA ATGCGGTATT GCTTGAACAA 
               
               
                   
               
               
                 421 
                 TTGAGAATCG GCGACCTAGA CTTGGTTGTG GGAAGGCTTG CCGCCCCGGA TAAAATGACT 
               
               
                   
               
               
                 481 
                 GGGTTTTCTT TCGAGCACCT ATACAGTGAG CAAGTTGTGT TTGCAGTAAG GGCAGGCCAT 
               
               
                   
               
               
                 541 
                 CCCCTGATCT CCGGTAGGCA ATCCTTGTTT GCTCATCTTT CCGACTACCC TGTTCTAATG 
               
               
                   
               
               
                 601 
                 CCAACAAGGG CCAGCATAAT TAGGCCATTC GTCGAGCACT TTTTGATAGC TAATGGCATC 
               
               
                   
               
               
                 661 
                 GCTGGTTTGC CAAACCAGAT AGAAACCGTC TCCGATTCAT TTGGTAGAGC TTTTGTACGT 
               
               
                   
               
               
                 721 
                 TCTTCCGACG CTATTTGGAT TATATCCGCT GGTGTAGTAG CTACTGATAT TGCCGATGGT 
               
               
                   
               
               
                 781 
                 GTTTTGGCAG CTCTACCAGT AGACACTTCA GAAACCCGTG GCCCTGTTGG CTTGACTATG 
               
               
                   
               
               
                 841 
                 AGAACCGATG CAATACCATC TTTGCCTCTT TCAATCTTAA TGCAAACTTT AAGAGAAGTG 
               
               
                   
               
               
                 901 
                 GCCGGTACCG CAATGGCAGC TGAAGCCAAA AGAACAGCAT AA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS ArgP 894 np DNA 
               
            
           
           
               
               
            
               
                   1 
                 ATGAAACGTC CTGATTATAG AACTCTGCAA GCCTTAGATG CTGTAATTAG AGAACGTGGC 
               
               
                   
               
               
                  61 
                 TTCGAGAGAG CGGCTCAGAA GTTGTGTATT ACTCAATCCG CCGTGAGCCA GAGAATAAAG 
               
               
                   
               
               
                 121 
                 CAGCTAGAAA ATATGTTTGG CCAACCATTA CTGGTACGTA CTGTTCCTCC TAGGCCGACG 
               
               
                   
               
               
                 181 
                 GAACAAGGTC AGAAGCTTTT GGCCTTGTTG AGACAAGTGG AGTTGCTAGA AGAGGAATGG 
               
               
                   
               
               
                 241 
                 TTGGGAGACG AGCAGACAGG TTCAACACCA CTTTTATTGA GTCTGGCCGT AAATGCGGAT 
               
               
                   
               
               
                 301 
                 AGCCTAGCTA CTTGGTTGCT ACCGGCTCTA GCTCCTGTCT TGGCTGACAG TCCCATAAGA 
               
               
                   
               
               
                 361 
                 TTAAACTTAC AAGTCGAAGA TGAAACGAGA ACGCAAGAAA GACTTAGGAG AGGAGAGGTC 
               
               
                   
               
               
                 421 
                 GTGGGGGCTG TATCAATTAA ACATAAGGCA TTGCCCAGTT GTATAGTAGA CAAGTTGGGT 
               
               
                   
               
               
                 481 
                 GCGCTAGATT ACCAATAAGT GTAATCCAAA CCTTTCGCCG AGAAGTATTA TCCTAATGGA 
               
               
                   
               
               
                 541 
                 GTAACCCGTT CCGCTTTGCT TAAAGCCCCA GTAGTAGCAT TCGACCATCT AGATGACATG 
               
               
                   
               
               
                 601 
                 CACCAAGCCT TTTTACAACA AAATTTCGAT TAACCACCAG GCTACGTACC ATGCCAAATC 
               
               
                   
               
               
                 661 
                 GTGAACTATA CCGAAGCCTA CGTACAACTA GCTAGTAAAG GAACTACTTG CTGTATGATT 
               
               
                   
               
               
                 721 
                 CCACATATAC AAATAGAAAA AGAATTGGCC TCCGGAGAAT TGATAGACCT GACACCTGGC 
               
               
                   
               
               
                 781 
                 CAATTAAAAA GaAGAAAGCT GAATTGGCAT AGGTTAGCAC CAGAGTCAAG AATGATGAGA 
               
               
                   
               
               
                 841 
                 AAGGTGACTG ATGCATTGCT TGATTATGGC CATAAGGTGT TAAGACAAGA TTGA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS MdcR 927 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGAAGGACG ACATCAATCA AGAAATTACC TTCAGGAAGT TATCTGTTTT CATGATGTTT 
               
               
                   
               
               
                  61 
                 ATGGCCAAAG GCAATATCGC CAGAACTGCT GAAGCAATGA AGTTATCATC TGTGTCAGTT 
               
               
                   
               
               
                 121 
                 CACAGAGCGC TGCATACACT AGAAGAAGGT GTGGGATGTC CCCTGTTCGT CCACAAAGGT 
               
               
                   
               
               
                 181 
                 AGAAATCTAC TACCTCTACA GGCAGCATGG ACTCTATTAG AATATTGCCA AGATGTAATT 
               
               
                   
               
               
                 241 
                 TCATTAATGA ATAGAGGACT AGAAGCCACT AGAAAAGTGG CAGGTGTTGG TCAAGGAAGA 
               
               
                   
               
               
                 301 
                 TTGAGAATCG GTACACTTTA CTCCTTAACA CTAGAAACCG TACCAAGGAT AATAATGGGC 
               
               
                   
               
               
                 361 
                 ATGAAGTTAA GACGTCCAGA ACTTGAGCTA GACTTGACAA TGGGTTCAAA TCAAATGTTA 
               
               
                   
               
               
                 421 
                 TTAGATATGC TAGAAGATGA TGCCTTAGAT GCAATATTGA TAGCTACCAA CGAAGGCGAA 
               
               
                   
               
               
                 481 
                 TTCAACAATA CTGCCTTTGA TGTTGTTCCT TTGTTTGAGG ATGACATATT TCTTGCAGCA 
               
               
                   
               
               
                 541 
                 CCTGCAACTG AACGTCTTGA CGCCTCAAGA TTGGCTGACC TGAGAGATTA CGCTGATAGA 
               
               
                   
               
               
                 601 
                 AAGTTTGTTT CCTTAGCGGA AGGATTTGCT ACCTATGCTG GTTTTCGTGA AGCTTTCCAT 
               
               
                   
               
               
                 661 
                 ATAGCTGGCT TTGAACCAGA GATAGTTACC AGAGTTAATG ACATATTCAG TATGATATCT 
               
               
                   
               
               
                 721 
                 CTTGTTCAGG CTGGTGTTGG GTTTGCTCTT TTGCCAGGAA GAATGAAGAA AGTTTATGAA 
               
               
                   
               
               
                 781 
                 AAGGACGTTC AATTGCTTAA GTTAGCCGAA CCTTACCAAA TGAGACAGCT GATTAGTATC 
               
               
                   
               
               
                 841 
                 GTATATTCCC ATCACAGGGA ACGTGACGCT GATTTGTTGG CATTAGCGGC TGAAGGTAGG 
               
               
                   
               
               
                 901 
                 ATGTATGCTC GTTCTATTAA CAGGTAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS 209 bp_CYC1p_BenO_T1::yeGFP 1014 bp 
               
            
           
           
               
               
            
               
                   1 
                 CCAGGCAACT TTAGTGCTGA CACATA ATAC TCCATAGGTA TTTTATTATA CAAATAATGT   
               
               
                   
               
               
                  61 
                   GTTTGAACTT ATTAAAACAT TCTTTTAAGG TATAAACAA C AGGCAAATAT ATATGTGTGC 
               
               
                   
               
               
                 121 
                 GACGACACAT GATAATATGG CATGCATGTG CTATGTATGT ATATAAAACT CTTGTATAAT 
               
               
                   
               
               
                 181 
                 TCTTTTATCT AAATATTCAA TACTAATACA TAAGGACCTT TGCAGCATAA ATTACTATAC 
               
               
                   
               
               
                 241 
                 TTCTAAAGAC ACACAAACAC AAATACACAC ACTAAATTAA TAATATGTAA TAAAACA ATG   
               
               
                   
               
               
                 301 
                 TATAAAGGTG AAGAATTATT CACTGGTGTT GTACCAATTA TGGTTGAATT AGATGGTGAT 
               
               
                   
               
               
                 361 
                 GTAAAAGGTA ACAAATTTTC TGTATACGGT GAAGGTGAAG GTGAAGCAAC TTACGGTAAA 
               
               
                   
               
               
                 421 
                 TTGACCTAAA AATTTATTTG TACTACTGGA AAATTGCCAG TTCCATGGCC AACCTTAGTC 
               
               
                   
               
               
                 481 
                 ACTACTAAAG GTAATGGTGT TCAATGTTAA GCGAGATACC CAGATAAAAT GAAACAACAT 
               
               
                   
               
               
                 541 
                 GACTAATTAA AGTATGCCAT GCCAGAAGGT TATGTTAAAG AAAGAACTAT TTTTTTCAAA 
               
               
                   
               
               
                 601 
                 GATGACGGAA ACTACAAGAC CAGAGCTGAA GAAAAGTATG AAGGTGATAC CTTAGTAAAT 
               
               
                   
               
               
                 661 
                 AGAATCGAAT TAAAAGGTAT TGATTTTAAA GAAGATGGTA ACATTATAGG TCACAAATTG 
               
               
                   
               
               
                 721 
                 GAATACAACT ATAACTATAA CAATGATAAC ATAATGGCTG ACAAACAAAA GAAAGGTAAA 
               
               
                   
               
               
                 781 
                 AAAGTTAACT TCAAAAATAG ACACAACAAA GAAGAAGGAT CAGTAAAATT AGCTGACCAT 
               
               
                   
               
               
                 841 
                 AATCAACAAA AAACTACAAT TGGTGATGGT CCAGAATAGT AACCAGACAA CCATTACTTA 
               
               
                   
               
               
                 901 
                 TCCACTCAAT CTGCCTAATC CAAAGAACCA AACGAAAAGA GAGACCACAA GGTCTTGATA 
               
               
                   
               
               
                 961 
                 GAATTTGAAA CTGCTGCTGG TATTACCCAT GGAATGGATG AATTGTACAA ATAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS 209bp_CYC1p_FdeO_T1::yeGFP 1014 bp 
               
            
           
           
               
               
            
               
                   1 
                 CCAGGCAACT TTAGTGCTGA CACATA AGCT TGATATTGAT CAAATGGATT GTTTTGATTC   
               
               
                   
               
               
                  61 
                   ATGATATGGA CGGCATCAAT ACATTGACCA CCCCATCCG C AGGCATATAT ATATGTGTGC 
               
               
                   
               
               
                 121 
                 GACGACACAT GATCATATGG CATGCATGTG CTCTGTATGT ATATAAAACT CTTGTTTTCT 
               
               
                   
               
               
                 181 
                 TCTTTTCTCT AAATATTCTT TCCTTATACA TTAGGACCTT TGCAGCATAA ATTACTATAC 
               
               
                   
               
               
                 241 
                 TTCTATAGAC ACACAAACAC AAATACACAC ACTAAATTAA TAATCTGTCA TAAAACA ATG   
               
               
                   
               
               
                 301 
                 TCTAAAGGTG AAGAATTATT CACTGGTGTT GTCCCAATTT TGGTTGAATT AGATGGTGAT 
               
               
                   
               
               
                 361 
                 GTTAATGGTC ACAAATTTTC TGTCTCCGGT GAAGGTGAAG GTGATGCTAC TTACGGTAAA 
               
               
                   
               
               
                 421 
                 TTGACCTTAA AATTTATTTG TACTACTGGT AAATTGCCAG TTCCATGGCC AACCTTAGTC 
               
               
                   
               
               
                 481 
                 ACTACTTTCG GTTATGGTGT TCAATGTTTT GCGAGATACC CAGATCATAT GAAACAACAT 
               
               
                   
               
               
                 541 
                 GACTTTTTCA AGTCTGCCAT GCCAGAAGGT TATGTTCAAG AAAGAACTAT TTTTTTCAAA 
               
               
                   
               
               
                 601 
                 GATGACGGTA ACTACAAGAC CAGAGCTGAA GTCAAGTTTG AAGGTGATAC CTTAGTTAAT 
               
               
                   
               
               
                 661 
                 AGAATCGAAT TAAAAGGTAT TGATTTTAAA GAAGATGGTA ACATTTTAGG TCACAAATTG 
               
               
                   
               
               
                 721 
                 GAATACAACT ATAACTCTCA CAATGTTTAC ATCATGGCTG ACAAACAAAA GAATGGTATC 
               
               
                   
               
               
                 781 
                 AAAGTTAACT TCAAAATTAG ACACAACATT GAAGATGGTT CTGTTCAATT AGCTGACCAT 
               
               
                   
               
               
                 841 
                 TATCAACAAA ATACTCCAAT TGGTGATGGT CCAGTCTTGT TACCAGACAA CCATTACTTA 
               
               
                   
               
               
                 901 
                 TCCACTCAAT CTGCCTTATC CAAAGATCCA AACGAAAAGA GAGACCACAT GGTCTTGTTA 
               
               
                   
               
               
                 961 
                 GAATTTGTTA CTGCTGCTGG TATTACCCAT GGTATGGATG AATTGTACAA ATAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS 209 bp_CYC1p_PcaO_T1::yeGFP 1021 bp 
               
            
           
           
               
               
            
               
                    1 
                 CCAGGCAACT TTAGTGCTGA CACATA GATC GTATAACCTC CTGGTTAAGG GAAAGCCACG   
               
               
                   
               
               
                   61 
                   AAATATCATT TTACCTAACC GGATGAAACA TCCAAATCTG ACGACG CAGG CATATATATA 
               
               
                   
               
               
                  121 
                 TGTGTGCGAC GACACATGAT CATATGGCAT GCATGTGCTC TGTATGTATA TAAAACTCTT 
               
               
                   
               
               
                  181 
                 GTTTTCTTCT TTTCTCTAAA TATTCTTTCC TTATACATTA GGACCTTTGC AGCATAAATT 
               
               
                   
               
               
                  241 
                 ACTATACTTC TATAGACACA CAAACACAAA TACACACACT AAATTAATAA TCTGTCATAA 
               
               
                   
               
               
                  301 
                 AACA ATG TCT AAAGGTGAAG AATTATTCAC TGGTGTTGTC CCAATTTTGG TTGAATTAGA 
               
               
                   
               
               
                  361 
                 TGGTGATGTT AATGGTCACA AATTTTCTGT CTCCGGTGAA GGTGAAGGTG ATGCTACTTA 
               
               
                   
               
               
                  421 
                 CGGTAAATTG ACCTTAAAAT TTATTTGTAC TACTGGTAAA TTGCCAGTTC CATGGCCAAC 
               
               
                   
               
               
                  481 
                 CTTAGTCACT ACTTTCGGTT ATGGTGTTCA ATGTTTTGCG AGATACCCAG ATCATATGAA 
               
               
                   
               
               
                  541 
                 ACAACATGAC TTTTTCAAGT CTGCCATGCC AGAAGGTTAT GTTCAAGAAA GAACTATTTT 
               
               
                   
               
               
                  601 
                 TTTCAAAGAT GACGGTAACT ACAAGACCAG AGCTGAAGTC AAGTTTGAAG GTGATACCTT 
               
               
                   
               
               
                  661 
                 AGTTAATAGA ATCGAATTAA AAGGTATTGA TTTTAAAGAA GATGGTAACA TTTTAGGTCA 
               
               
                   
               
               
                  721 
                 CAAATTGGAA TACAACTATA ACTCTCACAA TGTTTACATC ATGGCTGACA AACAAAAGAA 
               
               
                   
               
               
                  781 
                 TGGTATCAAA GTTAACTTCA AAATTAGACA CAACATTGAA GATGGTTCTG TTCAATTAGC 
               
               
                   
               
               
                  841 
                 TGACCATTAT CAACAAAATA CTCCAATTGG TGATGGTCCA GTCTTGTTAC CAGACAACCA 
               
               
                   
               
               
                  901 
                 TTACTTATCC ACTCAATCTG CCTTATCCAA AGATCCAAAC GAAAAGAGAG ACCACATGGT 
               
               
                   
               
               
                  961 
                 CTTGTTAGAA TTTGTTACTG CTGCTGGTAT TACCCATGGT ATGGATGAAT TGTACAAATA 
               
               
                   
               
               
                 1021 
                 A 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS 209bp_CYC1p_ArgO_T1::yeGEP 1028 bp 
               
            
           
           
               
               
            
               
                    1 
                 CCAGGCAACT TTAGTGCTGA CACATA TCTG GCCTCTCTCT TATTAGTTTT TCTGATTGCC   
               
               
                   
               
               
                   61 
                   AATTAATATT ATCAATTTCC GCTAATAACA ATCCCGCGAT ATAGTCTCTG CAT CAGGCAT 
               
               
                   
               
               
                  121 
                 ATATATATGT GTGCGACGAC ACATGATCAT ATGGCATGCA TGTGCTCTGT ATGTATATAA 
               
               
                   
               
               
                  181 
                 AACTCTTGTT TTCTTCTTTT CTCTAAATAT TCTTTCCTTA TACATTAGGA CCTTTGCAGC 
               
               
                   
               
               
                  241 
                 ATAAATTACT ATACTTCTAT AGACACACAA ACACAAATAC ACACACTAAA TTAATAATCT 
               
               
                   
               
               
                  301 
                 GTCATAAAAC A ATG TCTAAA GGTGAAGAAT TATTCACTGG TGTTGTCCCA ATTTTGGTTG 
               
               
                   
               
               
                  361 
                 AATTAGATGG TGATGTTAAT GGTCACAAAT TTTCTGTCTC CGGTGAAGGT GAAGGTGATG 
               
               
                   
               
               
                  421 
                 CTACTTACGG TAAATTGACC TTAAAATTTA TTTGTACTAC TGGTAAATTG CCAGTTCCAT 
               
               
                   
               
               
                  481 
                 GGCCAACCTT AGTCACTACT TTCGGTTATG GTGTTCAATG TTTTGCGAGA TACCCAGATC 
               
               
                   
               
               
                  541 
                 ATATGAAACA ACATGACTTT TTCAAGTCTG CCATGCCAGA AGGTTATGTT CAAGAAAGAA 
               
               
                   
               
               
                  601 
                 CTATTTTTTT CAAAGATGAC GGTAACTACA AGACCAGAGC TGAAGTCAAG TTTGAAGGTG 
               
               
                   
               
               
                  661 
                 ATACCTTAGT TAATAGAATC GAATTAAAAG GTATTGATTT TAAAGAAGAT GGTAACATTT 
               
               
                   
               
               
                  721 
                 TAGGTCACAA ATTGGAATAC AACTATAACT CTCACAATGT TTACATCATG GCTGACAAAC 
               
               
                   
               
               
                  781 
                 AAAAGAATGG TATCAAAGTT AACTTCAAAA TTAGACACAA CATTGAAGAT GGTTCTGTTC 
               
               
                   
               
               
                  841 
                 AATTAGCTGA CCATTATCAA CAAAATACTC CAATTGGTGA TGGTCCAGTC TTGTTACCAG 
               
               
                   
               
               
                  901 
                 ACAACCATTA CTTATCCACT CAATCTGCCT TATCCAAAGA TCCAAACGAA AAGAGAGACC 
               
               
                   
               
               
                  961 
                 ACATGGTCTT GTTAGAATTT GTTACTGCTG CTGGTATTAC CCATGGTATG GATGAATTGT 
               
               
                   
               
               
                 1021 
                 ACAAATAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS 209 bp_CYC1p_MdcO_T1::yeGFP 1030 bp 
               
            
           
           
               
               
            
               
                    1 
                 CCAGGCAACT TTAGTGCTGA CACATA ATCG TTACTCTGAT GCTAACGATC GGCCACCGCG   
               
               
                   
               
               
                   61 
                   CTTAATTGAT GCTCATAGCC TCGCGTCGCA CACTAATCTC CACCAGGACA AACAA CAGGC 
               
               
                   
               
               
                  121 
                 ATATATATAT GTGTGCGACG ACACATGATC ATATGGCATG CATGTGCTCT GTATGTATAT 
               
               
                   
               
               
                  181 
                 AAAACTCTTG TTTTCTTCTT TTCTCTAAAT ATTCTTTCCT TATACATTAG GACCTTTGCA 
               
               
                   
               
               
                  241 
                 GCATAAATTA CTATACTTCT ATAGACACAC AAACACAAAT ACACACACTA AATTAATAAT 
               
               
                   
               
               
                  301 
                 CTGTCATAAA ACA ATG TCTA AAGGTGAAGA ATTATTCACT GGTGTTGTCC CAATTTTGGT 
               
               
                   
               
               
                  361 
                 TGAATTAGAT GGTGATGTTA ATGGTCACAA ATTTTCTGTC TCCGGTGAAG GTGAAGGTGA 
               
               
                   
               
               
                  421 
                 TGCTACTTAC GGTAAATTGA CCTTAAAATT TATTTGTACT ACTGGTAAAT TGCCAGTTCC 
               
               
                   
               
               
                  481 
                 ATGGCCAACC TTAGTCACTA CTTTCGGTTA TGGTGTTCAA TGTTTTGCGA GATACCCAGA 
               
               
                   
               
               
                  541 
                 TCATATGAAA CAACATGACT TTTTCAAGTC TGCCATGCCA GAAGGTTATG TTCAAGAAAG 
               
               
                   
               
               
                  601 
                 AACTATTTTT TTCAAAGATG ACGGTAACTA CAAGACCAGA GCTGAAGTCA AGTTTGAAGG 
               
               
                   
               
               
                  661 
                 TGATACCTTA GTTAATAGAA TCGAATTAAA AGGTATTGAT TTTAAAGAAG ATGGTAACAT 
               
               
                   
               
               
                  721 
                 TTTAGGTCAC AAATTGGAAT ACAACTATAA CTCTCACAAT GTTTACATCA TGGCTGACAA 
               
               
                   
               
               
                  781 
                 ACAAAAGAAT GGTATCAAAG TTAACTTCAA AATTAGACAC AACATTGAAG ATGGTTCTGT 
               
               
                   
               
               
                  841 
                 TCAATTAGCT GACCATTATC AACAAAATAC TCCAATTGGT GATGGTCCAG TCTTGTTAGC 
               
               
                   
               
               
                  901 
                 AGACAACCAT TACTTATCCA CTCAATCTGC CTTATCCAAA GATCCAAACG AAAAGAGAGA 
               
               
                   
               
               
                  961 
                 CCACATGGTC TTGTTAGAAT TTGTTACTGC TGCTGGTATT ACCCATGGTA TGGATGAATT 
               
               
                   
               
               
                 1021 
                 GTACAAATAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS SpMAE1 1317 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGGGTGAAC TCAAGGAAAT CTTGAAACAG AGGTATCATG AGTTGCTTGA CTGGAATGTC 
               
               
                   
               
               
                   61 
                 AAAGCCCCTC ATGTCCCTCT CAGTCAACGA CTGAAGCATT TTACATGGTC TTGGTTTGCA 
               
               
                   
               
               
                  121 
                 TGTACTATGG CAACTGGTGG TGTTGGTTTG ATTATTGGTT CTTTCCCCTT TCGATTTTAT 
               
               
                   
               
               
                  181 
                 GGTCTTAATA CAATTGGCAA AATTGTTTAT ATTCTTCAAA TCTTTTTGTT TTCTCTCTTT 
               
               
                   
               
               
                  241 
                 GGATCATGCA TGCTTTTTCG CTTTATTAAA TATCCTTCAA CTATCAAGGA TTCCTGGAAC 
               
               
                   
               
               
                  301 
                 CATCATTTGG AAAAGCTTTT CATTGCTACT TGTCTTCTTT CAATATCCAC GTTCATCGAC 
               
               
                   
               
               
                  361 
                 ATGCTTGCCA TATACGCCTA TCCTGATACC GGCGAGTGGA TGGTGTGGGT CATTCGAATC 
               
               
                   
               
               
                  421 
                 CTTTATTACA ATTTTGTTGC AGTATCCTTT ATATACTGCG TAATGGCTTT TTTTACAATT 
               
               
                   
               
               
                  481 
                 TTCAACAACC ATGTATATAC CATTGAAACC GCATCTCCTG CTTGGATTCT TCCTATTTTC 
               
               
                   
               
               
                  541 
                 CCTCCTATGA TTTGTGGTGT CATTGCTGGC GCCGTCAATT CTACACAACC CGCTCATCAA 
               
               
                   
               
               
                  601 
                 TTAAAAAATA TGGTTATCTT TGGTATCCTC TTTCAAGGAC TTGGTTTTTG GGTTTATCTT 
               
               
                   
               
               
                  661 
                 AtTTTGTTTG CCGTCAATGT ATCTTGGTTT TTTACTGTAG GCCTGGCAAA ACCCCAAGAT 
               
               
                   
               
               
                  721 
                 CGACCTGGTA TGTTTATGTT TGTCGGTCCA CCAGCTTTCT CAGGTTTGGC CTTAATTAAT 
               
               
                   
               
               
                  781 
                 ATTGCGCGTG GTGCTATGGG CAGTCGCCCT TATATTTTTG TTGGCGCCAA CTCATCCGAG 
               
               
                   
               
               
                  841 
                 TATCTTGGTT TTGTTTCTAC CTTTATGGCT ATTTTTATTT GGGGTCTTGC TGCTTGGTGT 
               
               
                   
               
               
                  901 
                 TACTGTCTCG CCATGGTTAG CTTTTTAGCG GGCTTTTTCA CTCGAGCCCC TCTCAAGTTT 
               
               
                   
               
               
                  961 
                 GCTTGTGGAT GGTTTGCATT CATTTTCCCC AACGTGGGTT TTGTTAATTG TACCATTGAG 
               
               
                   
               
               
                 1021 
                 ATAGGTAAAA TGATAGATTC CAAAGCTTTC CAAATGTTTG GACATATCAT TGGGGTCATT 
               
               
                   
               
               
                 1081 
                 CTTTGTATTC AGTGGATCCT CCTAATGTAT TTAATGGTCC GTGCGTTTCT CGTCAATGAT 
               
               
                   
               
               
                 1141 
                 CTTTGCTATC CTGGCAAAGA CGAAGATGCC CATCCTCCAC CAAAACCAAA TACAGGTGTC 
               
               
                   
               
               
                 1201 
                 CTTAACCCTA CCTTCCCACC TGAAAAAGCA CCTGCATCTT TGGAAAAAGT CGATACACAT 
               
               
                   
               
               
                 1261 
                 GTCACATCTA CTGGTGGTGA ATCGGATCCT CCTAGTAGTG AACATGAAAG CGTTTAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS AtPAL-2 2154 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGGACCAAA TTGAAGCAAT GCTATGCGGT GGTGGTGAAA AGACCAAGGT GGCCGTAACG 
               
               
                   
               
               
                   61 
                 ACAAAAACTC TTGCAGATCC TTTGAATTGG GGTCTGGCAG CTGACCAGAT GAAAGGTAGC 
               
               
                   
               
               
                  121 
                 CATCTGGATG AAGTTAAGAA GATGGTTGAG GAATACAGAA GACCAGTCGT AAATCTAGGC 
               
               
                   
               
               
                  181 
                 GGCGAGACAT TGACGATAGG ACAGGTAGCT GCTATTTCGA CCGTTGGCGG TTCAGTGAAG 
               
               
                   
               
               
                  241 
                 GTAGAACTTG CAGAAACAAG TAGAGCCGGA GTTAAGGCTT CATCAGATTG GGTCATGGAA 
               
               
                   
               
               
                  301 
                 AGTATGAACA AGGGCACAGA TTCCTATGGC GTTACCACAG GCTTTGGTGC TACCTCTCAT 
               
               
                   
               
               
                  361 
                 AGAAGAACTA AAAATGGCAC TGCTTTGCAA ACAGAACTGA TCAGATTCCT TAACGCCGGT 
               
               
                   
               
               
                  421 
                 ATTTTCGGTA ATACAAAGGA AACTTGCCAT ACATTACCCC AATCGGCAAC AAGAGCTGCT 
               
               
                   
               
               
                  481 
                 ATGCTTGTTA GGGTGAACAC TTTGTTGCAA GGTTACTCTG GAATAAGGTT TGAAATTCTT 
               
               
                   
               
               
                  541 
                 GAGGCCATCA CTTCACTATT GAACCACAAC ATTTCTCCTT CGTTGCCCTT AAGAGGAACA 
               
               
                   
               
               
                  601 
                 ATAACTGCCA GCGGTGATTT GGTTCCCCTT TCATATATCG CAGGCTTATT AACGGGAAGA 
               
               
                   
               
               
                  661 
                 CCTAATTCAA AGGCCACTGG TCCAGACGGA GAATCCTTAA CCGCTAAGGA AGCATTTGAG 
               
               
                   
               
               
                  721 
                 AAAGCTGGTA TTTCAACTGG TTTCTTTGAT TTgCAACCCA AGGAAGGTTT AGCCCTGGTG 
               
               
                   
               
               
                  781 
                 AATGGCACCG CTGTCGGCAG CGGTATGGCA TCCATGGTGT TGTTTGAAGC TAACGTACAA 
               
               
                   
               
               
                  841 
                 GCAGTTTTGG CCGAAGTTTT GTCCGGAATT TTTGCCGAAG TCATGAGTGG AAAACCTGAG 
               
               
                   
               
               
                  901 
                 TTTACTGATC ACTTGACCCA CAGGTTAAAA CATCACCCAG GACAAATTGA AGCAGCAGCT 
               
               
                   
               
               
                  961 
                 ATCATGGAGC ACATTTTGGA CGGCTCTAGC TACATGAAGT TAGCCCAGAA GGTTCATGAA 
               
               
                   
               
               
                 1021 
                 ATGGACCCTT TGCAAAAACC CAAACAAGAT AGATATGCTT TAAGGACATC CCCACAATGG 
               
               
                   
               
               
                 1081 
                 CTTGGCCCTC AAATTGAAGT AATTAGACAA GCTACAAAGT CTATAGAAAG AGAGATCAAC 
               
               
                   
               
               
                 1141 
                 TCTGTTAACG ATAATCCACT TATTGATGTG TCGAGGAATA AGGCAATACA TGGAGGCAAT 
               
               
                   
               
               
                 1201 
                 TTCCAGGGTA CACCCATAGG AGTCAGTATG GATAATACCA GGCTTGCCAT AGCCGCAATT 
               
               
                   
               
               
                 1261 
                 GGCAAATTAA TGTTTGCCCA ATTTTCTGAA TTGGTCAATG ACTTCTACAA TAACGGTTTG 
               
               
                   
               
               
                 1321 
                 CCTTCGAATC TGACCGCATC TTCTAACCCT AGTCTTGATT ATGGTTTCAA AGGTGCTGAG 
               
               
                   
               
               
                 1381 
                 ATAGCAATGG CAAGCTATTG TTCAGAGCTG CAATATCTAG CCAACCCAGT AACCTCTCAT 
               
               
                   
               
               
                 1441 
                 GTACAATCAG CCGAACAACA CAATCAGGAT GTTAATTCTT TGGGCCTGAT TTCATCAAGA 
               
               
                   
               
               
                 1501 
                 AAAACAAGCG AGGCCGTTGA TATCCTTAAA TTAATGTCCA CAACATTTTT AGTGGGTATA 
               
               
                   
               
               
                 1561 
                 TGCCAGGCCG TAGATTTgAG ACACTTGGAA GAGAATTTGA GACAGACAGT GAAAAATACC 
               
               
                   
               
               
                 1621 
                 GTATCACAGG TTGCAAAAAA GGTTCTAACT ACAGGTATCA ATGGTGAATT GCACCCATCA 
               
               
                   
               
               
                 1681 
                 AGATTCTGTG AAAAAGATTT ATTAAAAGTT GTAGATAGAG AACAAGTATT TACTTACGTT 
               
               
                   
               
               
                 1741 
                 GACGATCCAT GTAGCGCTAC TTATCCATTG ATGCAGAGAT TGAGACAAGT TATTGTAGAT 
               
               
                   
               
               
                 1801 
                 CACGCTTTAT CCAATGGTGA AACTGAGAAA AATGCCGTTA CTTCAATATT CCAAAAGATA 
               
               
                   
               
               
                 1861 
                 GGTGCCTTTG AAGAAGAACT GAAGGCAGTT TTACCAAAGG AAGTCGAAGC TGCTAGAGCC 
               
               
                   
               
               
                 1921 
                 GCATACGGAA ATGGTACTGC CCCTATACCA AATAGAATCA AAGAGTGTAG GTCGTACCCT 
               
               
                   
               
               
                 1981 
                 TTGTACAGAT TCGTTAGAGA AGAGTTGGGA ACCAAATTAC TAACTGGTGA AAAAGTCGTT 
               
               
                   
               
               
                 2041 
                 AGCCCAGGTG AAGAATTTGA CAAGGTATTC ACAGCTATGT GCGAGGGAAA GTTGATAGAT 
               
               
                   
               
               
                 2101 
                 CCACTTATGG ATTGCTTGAA AGAGTGGAAT GGTGCACCTA TTCCAATCTG CTAA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS AtC4H::L5::AtATR2 3702 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGGATTTGT TATTGCTGGA AAAGTCACTT ATTGCTGTAT TTGTGGCAGT TATTCTAGCC 
               
               
                   
               
               
                   61 
                 ACGGTTATTT CTAAATTAAG AGGTAAGAAA CTAAAACTAC CTCCTGGTCC CATCCCCATA 
               
               
                   
               
               
                  121 
                 CCAATTTTTG GTAATTGGTT GCAAGTGGGC GATGATTTGA ATCACAGAAA TTTGGTAGAC 
               
               
                   
               
               
                  181 
                 TATGCTAAGA AGTTCGGTGA CCTTTTCTTG CTTAGAATGG GTCAAAGGAA TTTGGTAGTG 
               
               
                   
               
               
                  241 
                 GTTAGCTCAC CTGATTTGAC TAAGGAGGTC TTATTAACGC AAGGCGTTGA GTTTGGCTCC 
               
               
                   
               
               
                  301 
                 AGAACTAGAA ATGTTGTGTT TGATATTTTC ACTGGTAAAG GTCAAGATAT GGTTTTTACA 
               
               
                   
               
               
                  361 
                 GTTTACGGTG AGCACTGGAG AAAAATGAGA AGAATCATGA CCGTACCATT CTTTACTAAC 
               
               
                   
               
               
                  421 
                 AAGGTTGTTC AACAAAATAG AGAAGGTTGG GAGTTTGAGG CAGCTTCCGT AGTGGAAGAC 
               
               
                   
               
               
                  481 
                 GTAAAGAAAA ATCCAGATTC GGCCACAAAG GGTATAGTAC TAAGAAAAAG ACTACAATTG 
               
               
                   
               
               
                  541 
                 ATGATGTACA ACAATATGTT CAGAATTATG TTTGACAGAA GATTTGAAAG TGAAGATGAC 
               
               
                   
               
               
                  601 
                 CCTTTGTTCC TGAGACTTAA GGCTTTGAAT GGTGAAAGAT CGAGATTGGC TCAAAGTTTC 
               
               
                   
               
               
                  661 
                 GAATATAATT ACGGTGACTT TATTCCAATC TTAAGACCAT TTTTGAGAGG CTATTTGAAA 
               
               
                   
               
               
                  721 
                 ATTTGCCAAG ACGTCAAGGA TAGGAGGATC GCTCTTTTCA AGAAGTACTT TGTGGACGAG 
               
               
                   
               
               
                  781 
                 AGAAAGCAAA TAGCTTCTTC CAAGCCCACA GGTTCGGAAG GTTTAAAATG TGCAATTGAT 
               
               
                   
               
               
                  841 
                 CATATTTTAG AAGCTGAACA AAAAGGTGAA ATTAACGAAG ATAATGTTTT GTACATTGTA 
               
               
                   
               
               
                  901 
                 GAAAATATCA ATGTGGCTGC AATAGAAACA ACCTTATGGT CAATAGAATG GGGTATTGCT 
               
               
                   
               
               
                  961 
                 GAATTGGTGA ATCACCCAGA AATACAATCT AAACTGAGAA ACGAGCTAGA TACCGTTTTA 
               
               
                   
               
               
                 1021 
                 GGTCCAGGTG TCCAAGTTAC AGAACCTGAT TTGCATAAGT TACCCTACTT GCAAGCTGTG 
               
               
                   
               
               
                 1081 
                 GTTAAAGAAA CCTTGAGATT GAGAATGGCT ATTCCTCTTC TAGTTCCTCA TATGAACCTA 
               
               
                   
               
               
                 1141 
                 CATGATGCTA AACTGGCCGG TTATGATATT CCAGCAGAAA GTAAGATTTT AGTAAATGCA 
               
               
                   
               
               
                 1201 
                 TGGTGGTTGG CCAACAATCC AAACAGTTGG AAAAAGCCTG AAGAATTCAG ACCTGAAAGA 
               
               
                   
               
               
                 1261 
                 TTCTTCGAAG AGGAATCTCA TGTTGAAGCC AACGGAAATG ACTTCAGATA TGTACCTTTT 
               
               
                   
               
               
                 1321 
                 GGCGTTGGCA GAAGATCGTG TCCAGGAATA ATACTAGCCT TACCAATATT GGGTATCACA 
               
               
                   
               
               
                 1381 
                 ATTGGTAGGA TGGTTCAAAA TTTTGAGTTG CTACCACCAC CCGGACAATC GAAAGTCGAT 
               
               
                   
               
               
                 1441 
                 ACTTCAGAGA AAGGAGGACA ATTCTCATTG CATATTTTGA ATCATTCCAT TATAGTCATG 
               
               
                   
               
               
                 1501 
                 AAACCCAGAA ATTGTAGCGC TGAAGCTGCA GCAAAAGAAG CTGCAGCTAA AGAAGCTGCA 
               
               
                   
               
               
                 1561 
                 GCAAAAGCTT CCAGTAGCTC TTCCTCCTCA ACCTCGATGA TCGACTTAAT GGCTGCTATT 
               
               
                   
               
               
                 1621 
                 ATAAAAGGAG AACCAGTTAT AGTTAGTGAC CCTGCTAACG CAAGCGCTTA CGAATCCGTT 
               
               
                   
               
               
                 1681 
                 GCAGCCGAGT TGTCAAGTAT GCTTATAGAA AATAGACAGT TTGCTATGAT TGTAACGACC 
               
               
                   
               
               
                 1741 
                 AGCATCGCCG TTTTAATTGG TTGCATCGTG ATGTTGGTGT GGAGGAGGAG CGGTTCGGGC 
               
               
                   
               
               
                 1801 
                 AATTCAAAGA GGGTTGAACC ACTAAAGCCA TTAGTTATCA AACCTAGAGA AGAGGAAATT 
               
               
                   
               
               
                 1861 
                 GACGATGGAA GGAAGAAAGT CACTATATTC TTCGGCACCC AAACAGGTAC AGCTGAAGGT 
               
               
                   
               
               
                 1921 
                 TTTGCTAAGG CTCTAGGAGA AGAAGCAAAA GCTAGATATG AAAAGACGAG ATTCAAAATT 
               
               
                   
               
               
                 1981 
                 GTCGATCTGG ATGACTATGC CGCCGATGAT GACGAATACG AAGAAAAATT GAAGAAAGAA 
               
               
                   
               
               
                 2041 
                 GATGTCGCAT TTTTCTTCCT TGCCACCTAC GGCGACGGTG AACCAACAGA TAATGCCGCA 
               
               
                   
               
               
                 2101 
                 AGGTTTTACA AGTGGTTTAC TGAAGGTAAT GACAGAGGAG AATGGCTGAA GAATTTGAAA 
               
               
                   
               
               
                 2161 
                 TATGGTGTGT TCGGCCTTGG TAACAGACAG TACGAGCATT TTAATAAGGT CGCTAAGGTT 
               
               
                   
               
               
                 2221 
                 GTAGATGATA TACTTGTTGA ACAAGGTGCT CAAAGGTTAG TGCAGGTGGG CTTGGGTGAC 
               
               
                   
               
               
                 2281 
                 GATGATCAAT GTATTGAAGA TGACTTTACT GCTTGGAGAG AAGCCTTGTG GCCTGAATTA 
               
               
                   
               
               
                 2341 
                 GATACTATCC TTAGAGAAGA AGGTGACACT GCTGTTGCTA CCCCCTACAC TGCAGCAGTC 
               
               
                   
               
               
                 2401 
                 CTAGAATATA GAGTCTCAAT CCATGATTCA GAAGACGCCA AATTCAATGA TATTAACATG 
               
               
                   
               
               
                 2461 
                 GCCAACGGTA ACGGTTACAC CGTTTTTGAC GCACAACATC CATACAAAGC TAATGTTGCT 
               
               
                   
               
               
                 2521 
                 GTTAAAAGGG AACTTCACAC CCCAGAAAGT GACAGGTCAT GTATACATTT GGAATTTGAT 
               
               
                   
               
               
                 2581 
                 ATCGCTGGTA GTGGTTTGAC TTACGAAACA GGTGACCATG TCGGAGTACT TTGCGATAAT 
               
               
                   
               
               
                 2641 
                 TTGTCAGAAA CTGTTGATGA AGCTTTGAGG TTATTGGATA TGTCACCAGA TACTTACTTC 
               
               
                   
               
               
                 2701 
                 TCATTGCATG CAGAAAAAGA AGACGGAACT CCAATATCAA GCTCGCTTCC CCCTCCATTC 
               
               
                   
               
               
                 2761 
                 CCTCCCTGTA ACTTAAGAAC AGCCCTAACT AGATATGCTT GTTTACTGTC TTCTCCAAAG 
               
               
                   
               
               
                 2821 
                 AAAAGTGCTT TGGTTGCATT GGCAGCCCAC GCATCCGATC CTACCGAAGC TGAGAGATTA 
               
               
                   
               
               
                 2881 
                 AAGCATTTGG CTTCACCAGC CGGTAAAGAT GAATACAGTA AGTGGGTAGT GGAGAGCCAA 
               
               
                   
               
               
                 2941 
                 AGATCGCTTT TAGAAGTGAT GGCTGAGTTT CCAAGTGCTA AACCTCCTCT GGGTGTATTT 
               
               
                   
               
               
                 3001 
                 TTCGCTGGTG TGGCCCCAAG ATTGCAGCCT AGATTTTATT CCATATCCTC ATCTCCAAAA 
               
               
                   
               
               
                 3061 
                 ATTGCCGAAA CCAGAATTCA CGTGACATGT GCTCTGGTCT ACGAAAAGAT GCCAACAGGT 
               
               
                   
               
               
                 3121 
                 AGGATTCACA AGGGTGTCTG TTCTACCTGG ATGAAAAATG CTGTACCCTA TGAAAAATCC 
               
               
                   
               
               
                 3181 
                 GAAAATTGTT CTAGTGCACC AATTTTCGTA AGACAATCTA ATTTCAAGTT ACCAAGCGAT 
               
               
                   
               
               
                 3241 
                 TCTAAAGTAC CCATTATTAT GATCGGTCCA GGTACTGGTT TGGCCCCATT CAGAGGCTTC 
               
               
                   
               
               
                 3301 
                 TTGCAAGAAA GATTGGCTTT AGTGGAGAGT GGAGTTGAAT TGGGTCCTTC AGTTTTATTC 
               
               
                   
               
               
                 3361 
                 TTTGGTTGTA GAAACAGAAG AATGGACTTT ATCTACGAAG AAGAATTGCA GAGATTTGTT 
               
               
                   
               
               
                 3421 
                 GAAAGTGGTG CATTGGCCGA ATTGAGTGTT GCATTCAGCA GGGAAGGTCC AACCAAAGAA 
               
               
                   
               
               
                 3481 
                 TACGTTCAAC ACAAGATGAT GGACAAGGCT TCTGATATCT GGAATATGAT TTCCCAAGGT 
               
               
                   
               
               
                 3541 
                 GCTTATTTGT ATGTTTGTGG TGACGCTAAA GGAATGGCTA GAGATGTTCA TAGATCACTG 
               
               
                   
               
               
                 3601 
                 CATACAATCG CACAAGAACA AGGTAGCATG GATTCAACAA AAGCAGAGGG CTTTGTAAAG 
               
               
                   
               
               
                 3661 
                 AATCTTCAGA CAAGCGGTAG ATATCTGAGA GATGTATGGT AA 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS At4CL-2 1671 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGACGACAC AAGATGTGAT AGTCAATGAT CAGAATGATC AGAAACAGTG TAGTAATGAC 
               
               
                   
               
               
                   61 
                 GTCATTTTCC GATCGAGATT GCCTGATATA TACATCCCTA ACCACCTCCC ACTCCACGAC 
               
               
                   
               
               
                  121 
                 TACATCTTCG AAAATATCTC AGAGTTCGCC GCTAAGCCAT GCTTGATCAA CGGTCCCACC 
               
               
                   
               
               
                  181 
                 GGCGAAGTAT ACACCTACGC CGATGTCCAC GTAACATCTC GGAAACTCGC CGCCGGTCTT 
               
               
                   
               
               
                  241 
                 CATAACCTCG GCGTGAAGCA ACACGACGTT GTAATGATCC TCCTCCCGAA CTCTCCTGAA 
               
               
                   
               
               
                  301 
                 GTAGTCCTCA CTTTCCTTGC CGCCTCCTTC ATCGGCGCAA TCACCACCTC CGCGAACCCG 
               
               
                   
               
               
                  361 
                 TTCTTCACTC CGGCGGAGAT TTCTAAACAA GCCAAAGCCT CCGCTGCGAA ACTCATCGTC 
               
               
                   
               
               
                  421 
                 ACTCAATCCC GTTACGTCGA TAAAATCAAG AACCTCCAAA ACGACGGCGT TTTGATCGTC 
               
               
                   
               
               
                  481 
                 ACCACCGACT CCGACGCCAT CCCCGAAAAC TGCCTCCGTT TCTCCGAGTT AACTCAGTCC 
               
               
                   
               
               
                  541 
                 GAAGAACCAC GAGTGGACTC AATACCGGAG AAGATTTCGC CAGAAGACGT CGTGGCGCTT 
               
               
                   
               
               
                  601 
                 CCTTTCTCAT CCGGCACGAC GGGTCTCCCC AAAGGAGTGA TGCTAACACA CAAAGGTCTA 
               
               
                   
               
               
                  661 
                 GTCACGAGCG TGGCGCAGCA AGTCGACGGC GAGAATCCGA ATCTTTACTT CAACAGAGAC 
               
               
                   
               
               
                  721 
                 GACGTGATCC TCTGTGTCTT GCCTATGTTC CATATATACG CTCTCAACTC CATCATGCTC 
               
               
                   
               
               
                  781 
                 TGTAGTCTCA GAGTTGGTGC CACGATCTTG ATAATGCCTA AGTTCGAAAT CACTCTCTTG 
               
               
                   
               
               
                  841 
                 TTAGAGCAGA TACAAAGGTG TAAAGTCACG GTGGCTATGG TCGTGCCACC GATCGTTTTA 
               
               
                   
               
               
                  901 
                 GCTATCGCGA AGTCGCCGGA GACGGAGAAG TATGATCTGA GCTCGGTTAG GATGGTTAAG 
               
               
                   
               
               
                  961 
                 TCTGGAGCAG CTCCTCTTGG TAAGGAGCTT GAAGATGCTA TTAGTGCTAA GTTTCCTAAC 
               
               
                   
               
               
                 1021 
                 GCCAAGCTAG GTCAGGGCTA TGGGATGACA GAAGCAGGTC CGGTGCTAGC AATGTCGTTA 
               
               
                   
               
               
                 1081 
                 GGGTTTGCTA AAGAGCCGTT TCCAGTGAAG TCAGGAGCAT GTGGTACGGT GGTGAGGAAC 
               
               
                   
               
               
                 1141 
                 GCCGAGATGA AGATACTTGA TCCAGACACA GGAGATTCTT TGCCTAGGAA CAAACCCGGC 
               
               
                   
               
               
                 1201 
                 GAAATATGCA TCCGTGGCAA CCAAATCATG AAAGGCTATC TCAATGACCC CTTGGCCACG 
               
               
                   
               
               
                 1261 
                 GCATCGACGA TCGATAAAGA TGGTTGGCTT CACACTGGAG ACGTCGGATT TATCGATGAT 
               
               
                   
               
               
                 1321 
                 GACGACGAGC TTTTCATTGT GGATAGATTG AAAGAACTCA TCAAGTACAA AGGATTTCAA 
               
               
                   
               
               
                 1381 
                 GTGGCTCCAG CTGAGCTAGA GTCTCTCCTC ATAGGTCATC CAGAAATCAA TGATGTTGCT 
               
               
                   
               
               
                 1441 
                 GTCGTCGCCA TGAAGGAAGA AGATGCTGGT GAGGTTCCTG TTGCGTTTGT GGTGAGATCG 
               
               
                   
               
               
                 1501 
                 AAAGATTCAA ATATATCCGA AGATGAAATC AAGCAATTCG TGTCAAAACA GGTTGTGTTT 
               
               
                   
               
               
                 1561 
                 TATAAGAGAA TCAACAAAGT GTTCTTCACT GACTCTATTC CTAAAGCTCC ATCAGGGAAG 
               
               
                   
               
               
                 1621 
                 ATATTGAGGA AGGATCTAAG AGCAAGACTA GCAAATGGAT TAATGAACTA G 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS HaCHS 1173 bp 
               
            
           
           
               
               
            
               
                    1 
                 ATGGTTACTG TTGAAGAAGT TAGAAAAGCT CAAAGGGCAG AAGGTCCAGC CACAGTGATG 
               
               
                   
               
               
                   61 
                 GCTATTGGAA CCGCAGTTCC TCCAAATTGT GTAGATCAGG CCACTTATCC TGACTACTAC 
               
               
                   
               
               
                  121 
                 TTTAGAATAA CAAACTCTGA GCATAAGGCT GAATTGAAAG AAAAGTTCCA AAGGATGTGC 
               
               
                   
               
               
                  181 
                 GACAAATCAC AGATCAAGAA AAGATACATG TACCTTAATG AGGAAGTCCT AAAGGAAAAC 
               
               
                   
               
               
                  241 
                 CCAAATATGT GTGCATACAT GGCCCCTTCC CTTGACGCTA GACAAGATAT TGTGGTTGTA 
               
               
                   
               
               
                  301 
                 GAGGTCCCAA AATTGGGCAA GGAAGCAGCT GTTAAAGCCA TAAAGGAATG GGGTCAACCT 
               
               
                   
               
               
                  361 
                 AAGAGCAAAA TCACCCACCT TGTGTTTTGC ACTACAAGCG GAGTTGACAT GCCAGGCGCA 
               
               
                   
               
               
                  421 
                 GATTATCAGC TAACCAAACT TTTGGGTTTA AGGCCTTCTG TAAAAAGATT GATGATGTAC 
               
               
                   
               
               
                  481 
                 CAACAAGGTT GTTTCGCTGG AGGCACTGTC TTAAGACTAG CCAAGGATCT TGCAGAGAAC 
               
               
                   
               
               
                  541 
                 AACAAAGGTG CTAGGGTGTT GGTTGTATGC TCAGAAATTA CAGCCGTCAC CTTTAGAGGA 
               
               
                   
               
               
                  601 
                 CCAACTGACA CTCACTTAGA TTCCCTAGTT GGTCAGGCAT TGTTTGGCGA CGGTGCTGCC 
               
               
                   
               
               
                  661 
                 GCAATAATCA TTGGAAGTGA TCCTATTCCA GAGGTGGAAA AGCCTCTTTT TGAACTTGTT 
               
               
                   
               
               
                  721 
                 AGCGCTGCCC AAACTATATT GCCAGATTCT GAGGGTGCAA TCGACGGCCA CTTAAGGGAA 
               
               
                   
               
               
                  781 
                 GTAGGTCTAA CCTTCCATCT TTTGAAAGAT GTCCCTGGTT TAATTTCAAA GAACGTGGAA 
               
               
                   
               
               
                  841 
                 AAATCCCTAA CAGAGGCTTT TAAACCATTG GGTATAAGTG ACTGGAATAG CTTATTCTGG 
               
               
                   
               
               
                  901 
                 ATCGCTCACC CAGGCGGCCC TGCCATACTT GACCAGGTTG AAGCAAAATT GAGCTTAAAG 
               
               
                   
               
               
                  961 
                 CCAGAAAAAC TAAGAGCTAC TAGACATGTA TTGTCAGAGT ATGGTAACAT GTCCAGTGCC 
               
               
                   
               
               
                 1021 
                 TGTGTCCTTT TCATTTTGGA TGAAATGAGG AGAAAAAGCA AGGAGGACGG CCTAAAAACC 
               
               
                   
               
               
                 1081 
                 ACAGGTGAGG GAATCGAATG GGGTGTTCTA TTCGGCTTTG GTCCAGGCCT TACTGTGGAG 
               
               
                   
               
               
                 1141 
                 ACAGTTGTAC TTCATTCAGT CGCAATTAAT TAG 
               
               
                 // 
                   
               
               
                   
               
            
           
           
               
            
               
                 LOCUS PhCHI 726 bp 
               
            
           
           
               
               
            
               
                   1 
                 ATGTCTCCAC CAGTTTCTGT TACAAAAATG CAAGTCGAAA ATTATGCTTT TGCACCAACA 
               
               
                   
               
               
                  61 
                 GTGAACCCTG CCGGTTCCAC CAATACTTTG TTCTTAGCTG GAGCAGGCCA TAGAGGTCTA 
               
               
                   
               
               
                 121 
                 GAGATTGAAG GAAAGTTTGT GAAATTCACA GCCATAGGCG TATACCTTGA GGAAAGTGCT 
               
               
                   
               
               
                 181 
                 ATCCCATTTT TGGCAGAAAA GTGGAAAGGT AAGACCCCTC AGGAGTTAAC TGATAGCGTC 
               
               
                   
               
               
                 241 
                 GAGTTCTTTA ATGGGGTGGT TACAGGTCCA TTCGAAAAGT TTACCAGAGT AACTATGATT 
               
               
                   
               
               
                 301 
                 CTACCTCTTA CAGGAAAGCA ATATTCTGAG AAAGTCGCCG AAAACTGTGT TGCTCACTGG 
               
               
                   
               
               
                 361 
                 AAGGGCATAG GTACCTACAC TGATGACGAA GGAAGGGCAA TCGAGAAATT CTTGGATGTG 
               
               
                   
               
               
                 421 
                 TTTAGATCAG AAACATTCCC ACCTGGTGCT TCCATTATGT TTACTCAGAG TCCATTAGGC 
               
               
                   
               
               
                 481 
                 TTGTTAACCA TCAGCTTTGC CAAGGACGAT TCAGTTACCG GTACTGCAAA TGCTGTAATC 
               
               
                   
               
               
                 541 
                 GAGAACAAAC AACTATCAGA AGCCGTCCTT GAATCCATTA TTGGAAAGCA TGGTGTGAGT 
               
               
                   
               
               
                 601 
                 CCTGCAGCCA AATGCTCTGT TGCCGAGAGA GTAGCAGAAT TGTTAAAAAA GAGCTATGCT 
               
               
                   
               
               
                 661 
                 GAAGAGGCCT CAGTGTTCGG CAAACCAGAA ACCGAAAAGT CCACAATACC TGTTATCGGT 
               
               
                   
               
               
                 721 
                 GTGTAG 
               
               
                 // 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 List of oligonucleotides 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 CCM pathway 
                   
                   
               
               
                 genes and 
                   
                   
               
               
                 promoters 
                   
                   
               
               
                 Primer name 
                 Primer sequence, 5′ to 3′ 
               
               
                   
               
               
                 ID1564_PTEF1- 
                 CGTGCGAU 
                 Forward primer for USER cloning of the 
               
               
                 &gt;_U2_fw 
                 GCACACACCATAGCTTC 
                 TEF1 promoter 
               
               
                   
               
               
                 ID1565_PTEF1- 
                 ATGACAGAU 
                 Reverse primer for USER cloning of the 
               
               
                 &gt;_U2_rv 
                 TTGTAATTAAAACTTAG 
                 TEF1 promoter 
               
               
                   
               
               
                 ID3108_PTEF1_ 
                 AGCTACTGAU 
                 Forward primer for USER cloning of the 
               
               
                 for_fusion_fw 
                 GCACACACCATAGCTTC 
                 TEF1 promoter fused with the TDH3 
               
               
                   
                   
                 promoter for bidirectional expression 
               
               
                   
               
               
                 ID3107_PTDH3_ 
                 ATCAGTAGCU 
                 Forward primer for USER cloning of the 
               
               
                 for_fusion_fw 
                 ATAAAAAACACGCTTTTTCAG 
                 TDH3 promoter fused with the TEF1 
               
               
                   
                   
                 promoter for bidirectional expression 
               
               
                   
               
               
                 ID1853_PTDH3 
                 ACCTGCACU 
                 Reverse primer for USER cloning of the 
               
               
                 &lt;-_U1_rv 
                 TTTGTTTGTTTATGTGTGTTTATT 
                 TDH3 promoter 
               
               
                   
                 C 
                   
               
               
                   
               
               
                 ID3097_CaCatA_ 
                 ATCTGTCAU 
                 Forward primer for USER cloning 
               
               
                 U2_fw 
                 AAAACAATGTCCCAAG 
                 of CaCatA 
               
               
                   
               
               
                 ID3098_CaCatA_ 
                 CACGCGAU 
                 Reverse primer for USER cloning 
               
               
                 U2_rv 
                 TTACAACTTGATTTCAGC 
                 of CaCatA 
               
               
                   
               
               
                 ID3103_KpAroY. 
                 ATCTGTCAU 
                 Forward primer for USER cloning 
               
               
                 B_U1_fw 
                 AAAACAATGATCTGTCC 
                 of KpAroY.B 
               
               
                   
               
               
                 ID3104_KpAroY. 
                 CGTGCGAU 
                 Reverse primer for USER cloning 
               
               
                 B_U1_rv 
                 TCATTCAATTTCTTGAGC 
                 of KpAroY.B 
               
               
                   
               
               
                 ID3105_KpAroY. 
                 ATCTGTCAU 
                 Forward primer for USER cloning 
               
               
                 Ciso_U2_fw 
                 AAAACAATGACCGCCCCAATC 
                 of KpAroY.Ciso 
               
               
                   
               
               
                 ID3016_KpAroY. 
                 CACGCGAU 
                 Reverse primer for USER cloning of 
               
               
                 Ciso_U2_rv 
                 TTACTTAGCGGAACCTTGATTC 
                 KpAroY.Ciso 
               
               
                   
               
               
                 ID3095_KpAroY. 
                 ATCTGTCAU 
                 Forward primer for USER cloning 
               
               
                 D_U2_fw 
                 AAAACAATGATCTGTCC 
                 of KpAroY.D 
               
               
                   
               
               
                 ID3096_KpAroY. 
                 CACGCGAU 
                 Reverse primer for USER cloning of 
               
               
                 D_U2_rv 
                 TTATCTCTTATCTTCTGG 
                 KpAroY.D 
               
               
                   
               
               
                 ID3101_PaAroZ_ 
                 AGTGCAGGU 
                 Forward primer for USER cloning 
               
               
                 U1_fw 
                 AAAACAATGCCATCCAAG 
                 of PaAroZ 
               
               
                   
               
               
                 ID3102_PaAroZ_ 
                 CGTGCGAU 
                 Reverse primer for USER cloning of 
               
               
                 U1_rv 
                 TCACAAAGCAGCTGACAAAG 
                 PaAroZ 
               
               
                   
               
               
                 ID1391_ScTkl1_ 
                 AGTGCAGGU 
                 Forward primer for USER cloning of Tkl1 
               
               
                 U1_fw 
                 AAAACAATGACTCAATTCACTGA 
                   
               
               
                   
                 CATTG 
                   
               
               
                   
               
               
                 ID1392_ScTkl1_ 
                 CGTGCGAU 
                 Reverse primer for USER cloning of Tkl1 
               
               
                 U1_rv 
                 TCAGAAAGCTTTTTTCAAAGGAG 
               
               
                   
               
            
           
           
               
               
            
               
                 LTTR sensor and reporter promoters 
                   
               
            
           
           
               
               
               
            
               
                 Primer name 
                 Primer sequence, 5′ to 3′ 
                   
               
               
                   
               
               
                 MeLS069-F 
                 GATGAATGCGGCCGCTTTA 
                 Forward primer for random mutagenesis 
               
               
                   
                   
                 of BenM-EBD 
               
               
                   
               
               
                 MeLS093-R 
                 CAATACGCCATCAAGTTGCTAAG 
                 Reverse primer for random mutagenesis 
               
               
                   
                 C 
                 of BenM-EBD 
               
               
                   
               
               
                 MeLS071-F 
                 CTCCTTCCTTTTCGGTTAGAGCG 
                 Tailed primer for BenM-EBD library 
               
               
                   
                 GATGAATGCGGCCGCTTTA 
                 assembly by gap repair 
               
               
                   
               
               
                 MeLS094-R 
                 TCATTTCTTTTACCAATACGCCAT 
                 Tailed primer for BenM-EBD library 
               
               
                   
                 CAAGTTGCTAAGC 
                 assembly by gap repair 
               
               
                   
               
               
                 MeLS001_F 
                 ATCTGTCAUAAAACAATGGAATT 
                 Forward primer for USER cloning of BenM 
               
               
                   
                 GAGACAC 
                   
               
               
                   
               
               
                 MeLS003_R 
                 CACGCGAUTTACCAATTTGGTGG 
                 Reverse primer for USER cloning of BenM 
               
               
                   
                 TTCAG 
                   
               
               
                   
               
               
                 MeLS005_F 
                 CGTGCGAUATACTCCATAGGTAT 
                 Forward primer for USER cloning of the 
               
               
                   
                 TTT 
                 BenM binding site 
               
               
                   
               
               
                 MeLS008_R 
                 CACGCGAUTTATTTGTACAATTC 
                 Reverse primer for USER cloning of the 
               
               
                   
                 ATCCA 
                 yeGFP ORF 
               
               
                   
               
               
                 MeLS009_F 
                 ATCTGTCAUAAAACAATGTCTAA 
                 Forward primer for USER cloning of the 
               
               
                   
                 AGGTG 
                 yeGFP ORF 
               
               
                   
               
               
                 MeLS0046_F 
                 CGTGCGAUTTCTTAGGCACAACA 
                 Forward primer for USER cloning of the 
               
               
                   
                 ATATTTATAAAAGAAG 
                 REV1 promoter 
               
               
                   
               
               
                 MeLS0047_R 
                 ATGACAGAUCGCTGGATATGCC 
                 Reverse primer for USER cloning of the 
               
               
                   
                 TAGAAATGC 
                 REV1 promoter 
               
               
                   
               
               
                 MeLS0048_F 
                 CGTGCGAUGGAAAACCAAGAAA 
                 Forward primer for USER cloning of the 
               
               
                   
                 TGAATTATATTTCC 
                 491 bp CYC1 promoter 
               
               
                   
               
               
                 MeLS0049_R 
                 ATGACAGAUTATTAATTTAGTGT 
                 Reverse primer for USER cloning of the 
               
               
                   
                 GTGTATTTGTGTTTGTG 
                 CYC1 promoter 
               
               
                   
               
               
                 MeLS0052_F 
                 CGTGCGAUCCAGGCAACTTTAG 
                 Forward primer for USER cloning of the 
               
               
                   
                 TGCTGACAC 
                 209 bp CYC1 promoter 
               
               
                   
               
               
                 MeLS0056_F 
                 CGTGCGAUATAAAAAACACGCTT 
                 Forward primer for USER cloning of the 
               
               
                   
                 TTTCAGTTCG 
                 TDH3 promoter 
               
               
                   
               
               
                 MeLS0057_R 
                 ATGACAGAUTTTGTTTGTTTATG 
                 Reverse primer for USER cloning of the 
               
               
                   
                 TGTGTTTATTCGA 
                 TDH3 promoter 
               
               
                   
               
               
                 MeLS0062_F 
                 CGTGCGAUGAAAGACCACACCC 
                 Forward primer for USER cloning of the 
               
               
                   
                 ACGCG 
                 RNR2 promoter 
               
               
                   
               
               
                 MeLS0063_R 
                 ATGACAGAUGGTAATTGGACAA 
                 Reverse primer for USER cloning of the 
               
               
                   
                 ATAAATACGTGTATTAAG 
                 RNR2 promoter 
               
               
                   
               
               
                 MeLS0064_F 
                 CGTGCGAUTCAAGCCCACGCGT 
                 Forward primer for USER cloning of the 
               
               
                   
                 AGGC 
                 272 bp CYC1 promoter 
               
               
                   
               
               
                 MeLS0074_F 
                 CGTGCGAUTCGAGCAGATCCGC 
                 Forward primer for USER cloning of the 
               
               
                   
                 CAGG 
                 249 bp CYC1 promoter 
               
               
                   
               
               
                 MelS101-R 
                 CACGCGAUTCAGCAGATGCCTG 
                 Reverse primer for USER cloning of FdeR 
               
               
                   
                 GCAGC 
                   
               
               
                   
               
               
                 MelS108-F 
                 ATCTGTCAUAAAACAATGCGTTT 
                 Forward primer for USER cloning of FdeR 
               
               
                   
                 CAACAAGCTCGAC 
                   
               
               
                   
               
               
                 TISNO-53F 
                 ATCTGTCAUAAAACAATGATTGA 
                 Forward primer for USER cloning of PcaQ 
               
               
                   
                 TGCACGT 
                   
               
               
                   
               
               
                 TISNO-54R 
                 CACGCGAUTTATGCTGTTCTTTT 
                 Reverse primer for USER cloning of PcaQ 
               
               
                   
                 GGCTTC 
                   
               
               
                   
               
               
                 TISNO-57F 
                 ATCTGTCAUAAAACAATGAAACG 
                 Forward primer for USER cloning of ArgP 
               
               
                   
                 TCCTGA 
                   
               
               
                   
               
               
                 TISNO-58R 
                 CACGCGAUTCAATCTTGTCTTAA 
                 Reverse primer for USER cloning of ArgP 
               
               
                   
                 CACCTTATG 
                   
               
               
                   
               
               
                 TISNO-59F 
                 ATCTGTCAUAAAACAATGAAGGA 
                 Forward primer for USER cloning of MdcR 
               
               
                   
                 CGACAT 
                   
               
               
                   
               
               
                 TISNO-60R 
                 CACGCGAUTTACCTGTTAATAGA 
                 Reverse primer for USER cloning of MdcR 
               
               
                   
                 ACGAGCATA 
               
               
                   
               
               
                 Genotyping 
                   
                   
               
               
                 primers 
                   
                   
               
               
                 Primer name 
                 Primer sequence, 5′ to 3′ 
               
               
                   
               
               
                 ID893 XII-2- 
                 CGAAGAAGGCCTGCAATTC 
                 Genotyping of genomic integration locus 
               
               
                 up-out-sq 
                   
                   
               
               
                   
               
               
                 ID894 XII-2- 
                 GGCCCTGATAAGGTTGTTG 
                 Genotyping of genomic integration locus 
               
               
                 down-out-sq 
                   
                   
               
               
                   
               
               
                 ID897 XII-4- 
                 GAACTGACGTCGAAGGCTCT 
                 Genotyping of genomic integration locus 
               
               
                 out-seq_fw 
                   
                   
               
               
                   
               
               
                 ID898 XII-4- 
                 CGTGAAATCTCTTTGCGGTAG 
                 Genotyping of genomic integration locus 
               
               
                 down-out-sq 
                   
                   
               
               
                   
               
               
                 ID903_X-3- 
                 TGACGAATCGTTAGGCACAG 
                 Genotyping of genomic integration locus 
               
               
                 out-seq_fw 
                   
                   
               
               
                   
               
               
                 ID904_X-3- 
                 CCGTGCAATACCAAAATCG 
                 Genotyping of genomic integration locus 
               
               
                 down-out-sq 
                   
                   
               
               
                   
               
               
                 ID905_X-4- 
                 CTCACAAAGGGACGAATCCT 
                 Genotyping of genomic integration locus 
               
               
                 out-seq_fw 
                   
                   
               
               
                   
               
               
                 ID906_X-4- 
                 GACGGTACGTTGACCAGAG 
                 Genotyping of genomic integration locus 
               
               
                 down-out-sq 
                   
                   
               
               
                   
               
               
                 ID907_XI-1- 
                 CTTAATGGGTAGTGCTTGACACG 
                 Genotyping of genomic integration locus 
               
               
                 out-seq_fw 
                   
                   
               
               
                   
               
               
                 ID908_XI-1- 
                 GAAGACCCATGGTTCCAAGGA 
                 Genotyping of genomic integration locus 
               
               
                 down-out-sq 
                   
                   
               
               
                   
               
               
                 ID911_XI-3- 
                 GTGCTTGATTTGCGTCATTC 
                 Genotyping of genomic integration locus 
               
               
                 out-seq_fw 
                   
                   
               
               
                   
               
               
                 ID2220 
                 CCTGCAGGACTAGTGCTGAG 
                 Genotyping of genomic integration locus 
               
               
                 Sc_ColoPCR_fw 
                   
                   
               
               
                   
               
               
                 ID2221 
                 GTTGACACTTCTAAATAAGCGAA 
                 Genotyping of genomic integration locus 
               
               
                 Sc_ColoPCR_rv 
                 TTTC 
                   
               
               
                   
               
               
                 MeLS0082_F 
                 AAAAATAAATAGGGACCTAGACT 
                 Sequencing primer located in the CYC1 
               
               
                   
                 TCAGG 
                 terminator 
               
               
                   
               
               
                 MeLS0053_R 
                 CTGCAGGAATTCGATATCAAGC 
                 Reverse sequencing primer located in the 
               
               
                   
                   
                 KI.URA terminator 
               
               
                   
               
               
                 MeLS0054_F 
                 TCAATTGAGATGAGCTTAATCAT 
                 Forward sequencing primer located in the 
               
               
                   
                 GTC 
                 KI.URA promoter 
               
               
                   
               
               
                 MeLS0055_R 
                 ATTATTACAGTCACTCAGACAGA 
                 Sequencing primer located in the XII-1 
               
               
                   
                 GCAC 
                 down homology region 
               
               
                   
               
               
                 MeLS0058_F 
                 GTGAAGTGATCATGCACATCGC 
                 Sequencing primer located in the REV1 
               
               
                   
                   
                 promoter 
               
               
                   
               
               
                 MeLS0083_R 
                 GAGGTTCCAGACCAGTTAAGACT 
                 Sequencing primer located in BenM DNA- 
               
               
                   
                 ACTC 
                 binding domain 
               
               
                   
               
               
                 TISNO-15F 
                 GTAAGCCAGATTAAAATTCACG 
                 Sequencing primer located in REV1 
               
               
                   
                   
                 promoter 
               
               
                   
               
               
                 TISNO-62R 
                 TAGCATCACCTTCACCTTCACC 
                 Genotyping of genomic integration locus 
               
               
                   
                   
                 (anneals to yeGFP) 
               
               
                   
               
               
                 TISNO-63F 
                 CCTGAAATTATTCCCCTACTTGA 
                 Sequencing primer located in TDH3 
               
               
                   
                 C 
                 promoter 
               
               
                   
               
               
                 TISNO-65R 
                 TATCGGATAACAACACCGCTG 
                 Genotyping of genomic integration locus 
               
               
                   
                   
                 (anneals to REV1p) 
               
               
                   
               
               
                 TISNO-66R 
                 CTGTTCACCCAGACACCTAC 
                 Genotyping of genomic integration locus 
               
               
                   
                   
                 (anneals to TDH3p) 
               
               
                   
               
               
                 TISNO-67R 
                 GCGGAGTCCGAGAAAATCTG 
                 Genotyping of genomic integration locus 
               
               
                   
                   
                 (anneals to TEF1p) 
               
               
                   
               
               
                 Res417 R 
                 TCTCAGGTATAGCATGAGGTCGC 
                 Genotyping of genomic integration locus, 
               
               
                   
                 TCAT 
                 internal assembler primer 
               
               
                   
               
               
                 Res418 F 
                 CCTGCAGGACTAGTGCTGAGGC 
                 Genotyping of genomic integration locus, 
               
               
                   
                 ATTAAT 
                 internal assembler primer 
               
               
                   
               
               
                 RE5395 XI-2 
                 GTTTGTAGTTGGCGGTGGAG 
                 Genotyping of genomic integration locus 
               
               
                 UP F 
                   
                   
               
               
                   
               
               
                 RE5396 XI-2 
                 GAGACAAGATGGGGCAAGAC 
                 Genotyping of genomic integration locus 
               
               
                 DW  
                   
                   
               
               
                   
               
               
                 RES511 XVI-20 
                 GGCTTGTGGTCACCTGTCAT 
                 Genotyping of genomic integration locus 
               
               
                 UP F 
                   
                   
               
               
                   
               
               
                 RE5512 XVI-20 
                 GAATTATGGTAATTTTGATTATC 
                 Genotyping of genomic integration locus 
               
               
                 DW R 
                   
                   
               
               
                   
               
               
                 RE5658 X-2-UP 
                 TGCGACAGAAGAAAGGGAAG 
                 Genotyping of genomic integration locus 
               
               
                 F 
                   
                   
               
               
                   
               
               
                 RE5659 X-2 
                 GAGAACGAGAGGACCCAACAT 
                 Genotyping of genomic integration locus 
               
               
                 DW R 
               
               
                   
               
            
           
           
               
               
            
               
                 Naringenin pathway genes and promoters 
                   
               
            
           
           
               
               
               
            
               
                 Primer name 
                 Primer sequence, 5′ to 3′ 
                   
               
               
                   
               
               
                 RES194 C4H F 
                 AGCGATACGUAAAATGGATTTGT 
                 Forward primer for USER cloning of the 
               
               
                   
                 TATTGCTGGAAAAG 
                 C4H 
               
               
                   
               
               
                 RES195 C4H R 
                 CACGCGAUTTACCATACATCTCT 
                 Reverse primer for USER cloning of the 
               
               
                   
                 CAGATATCTAC 
                 C4H 
               
               
                   
               
               
                 RE5196 AtPAL2 
                 ATCAACGGGUAAAATGGACCAA 
                 Forward primer for USER cloning of the 
               
               
                 F 
                 ATTGAAGCAATGC 
                 AtPAL2 
               
               
                   
               
               
                 RE5197 AtPAL2 
                 CGTGCGAUTTAGCAGATTGGAA 
                 Reverse primer for USER cloning of the 
               
               
                 R 
                 TAGGTGCAC 
                 AtPAL2 
               
               
                   
               
               
                 RE5198 At4CL2 
                 AGCGATACGUAAAATGACGACA 
                 Forward primer for USER cloning of the 
               
               
                 F 
                 CAAGATGTGATAGTC 
                 4Cl2 
               
               
                   
               
               
                 RES199 At4CL2 
                 CACGCGAUCTAGTTCATTAATCC 
                 Reverse primer for USER cloning of the 
               
               
                 R 
                 ATTTGCTAG 
                 4Cl2 
               
               
                   
               
               
                 RE5569 CHI F 
                 AGCGATACGUAAAATGTCTCCAC 
                 Forward primer for USER cloning of the 
               
               
                   
                 CAGTTTCTGTTAC 
                 CHI 
               
               
                   
               
               
                 RE5570 CHI R 
                 CACGCGAUCTACACACCGATAA 
                 Reverse primer for USER cloning of the 
               
               
                   
                 CAGGTATTG 
                 CHI 
               
               
                   
               
               
                 RE5571 CHS F 
                 CGTGCGAUTAATTAATTGCGACT 
                 Forward primer for USER cloning of the 
               
               
                   
                 GAATGAAG 
                 CHS 
               
               
                   
               
               
                 RE5572 CHS R 
                 ATCAACGGGUAAAATGGTTACTG 
                 Reverse primer for USER cloning of the 
               
               
                   
                 TTGAAGAAGTTAG 
                 CHS 
               
               
                   
               
               
                 RE5573 ATR2 
                 agctgcagcUaaagaagctgcagcaaa 
                 Forward primer for USER cloning of the 
               
               
                 L5 F 
                 agctTCCAGTAGCTCTTCCTCCTC 
                 ATR2 (includes L5 linker) 
               
               
                   
               
               
                 RES 574 L5 
                 AgctgcagcUtcttttgctgcagcttcagc 
                 Reverse primer for USER cloning of the 
               
               
                 C4H R 
                 gctACAATTTCTGGGTTTCATG 
                 C4H (includes L5 linker) 
               
               
                   
               
               
                 RE5407 pTDH3 
                 ACCCGTTGAUTTTTGTTTGTTTAT 
                 Forward primer for USER cloning of the 
               
               
                 F 
                 GTGTGTTTATTCG 
                 TDH3 promoter 
               
               
                   
               
               
                 RE5408 pTDH3 
                 CACGCGAUGATCTCAGTTCGAG 
                 Reverse primer for USER cloning of the 
               
               
                 R 
                 TTTATCATTATCA 
                 TDH3 promoter 
               
               
                   
               
               
                 RE5454 pPGK1 
                 ACCCGTTGAUGCCGCTTGTTTTA 
                 Reverse primer for USER cloning of the 
               
               
                 R 
                 TATTTGTTGTAAAAAG 
                 PGK1 promoter 
               
               
                   
               
               
                 RE5455 pPGK1 
                 CACGCGAUGGCCTGGAAGTACC 
                 Forward primer for USER cloning of the 
               
               
                 F 
                 TTCAAAGAATG 
                 PGK1 promoter 
               
               
                   
               
               
                 RE5456 pTEF1 
                 CGTGCGAUGCCGCACACACCAT 
                 Forward primer for USER cloning of the 
               
               
                 F 
                 AGCTTCAAAATG 
                 TEF1 promoter 
               
               
                   
               
               
                 RE5457 pTEF1 
                 ACGTATCGCUGTGAGTCGTATTA 
                 Reverse primer for USER cloning of the 
               
               
                 R 
                 CGGATCC 
                 TEF1 promoter 
               
               
                   
               
               
                 RE5568 pPDC1 
                 CGTGCGAUGCCGATCTATGCGA 
                 Forward primer for USER cloning of the 
               
               
                 F 
                 CTGGGTGAG 
                 PDC1 promoter 
               
               
                   
               
               
                 RE5640 pPDC1 
                 ACGTATCGCUTTTTGATAGATTT 
                 Reverse primer for USER cloning of the 
               
               
                 R 
                 GACTGTGTTATTTTGCG 
                 PDC1 promoter 
               
               
                   
               
               
                 RE5460 
                 ACCCGTTGAUTTTTGTTTGTTTAT 
                 Reverse primer for USER cloning of the 
               
               
                 pTDH3/pTEF2 
                 GTGTG 
                 bidirectional promoter 
               
               
                 R 
                   
                   
               
               
                   
               
               
                 RE5461 pTDH3 
                 ACGTATCGCUTGTTTAGTTAATT 
                 Forward primer for USER cloning of the 
               
               
                 /pTEF2 F 
                 ATAGTTC 
                 bidirectional promoter 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
             
            
               
                 AlsR 
                 Activator (local) 
                 
                   Bacillus subtilis 
                 
                 Renna et al. (1993) Bartowsky 
               
               
                 AmpR 
                 Activator (local) 
                 
                   Rhodobacter 
                 
                 &amp; Normark (1993) 
               
               
                   
                 Activator (local) 
                 
                   capsulatus 
                 
               
               
                 ArgP 
                 Activator (global) 
                 
                   Enterobacter cloacae 
                 
                 Nandineni &amp; Gowrishankar 
               
               
                 BenM 
                 Activator (local) 
                 
                   Citrobacter freundii 
                 
                 (2004) Collier et al. (1998) 
               
               
                 BlaA 
                 Activator (global) 
                 
                   Escherichia 
                 
                 Raskin et al. (2003) 
               
               
                 CatM 
                 Activator (global) 
                   Acinetobacter  spp. 
                 Chugani et al. (1998) van 
               
               
                 CbbR 
                 Activator (global) 
                   Streptomyces  spp. 
                 Keulen et al. (2003) 
               
               
                 CfxR 
                   
                 
                   Acinetobacter 
                 
                 Windho {umlaut over ( )}vel 991) 
               
               
                   
                   
                 
                   calcolaceticus 
                 
               
               
                   
                   
                 
                   Pseudomonas putida 
                 
               
               
                   
                   
                 
                   Xanthobacter flavus 
                 
               
               
                 ChiR 
                 Activator (local) 
                 
                   Serratia marcescens 
                 
                 Suzuki et al. (2001) 
               
               
                 CidR 
                 Activator (local) 
                   Staphylococcus  Spp. 
                 Yang et al. (2005) 
               
               
                   
                   
                 
                   Bacillus anthracis 
                 
                 Ahn et al. (2006) 
               
               
                 ClcR 
                 Activator (local) 
                 
                   Pseudomonas putida 
                 
                 Coco et al. (1993) 
               
               
                 CrqA 
                 Activator/Repressor 
                 
                   Neisseria meningitidis 
                 
                 Deghmane et al. (2000) 
               
               
                   
                 (global) 
               
               
                 CynR 
                 Activator (local) 
                 
                   Escherichia coli 
                 
                 Sung &amp; Fuchs (1992) 
               
               
                 CysB 
                 Activator (global) 
                 
                   Salmonella enterica 
                 
                 van der Ploeg et al. (1997) 
               
               
                   
                   
                 
                   Typhimurium 
                 
               
               
                   
                   
                 
                   Escherichia coli 
                 
               
               
                 CysL 
                 Activator (global) 
                 
                   Bacillus subtilis 
                 
                 Guillouard et al. (2002) 
               
               
                 GltC 
                 Activator (local) 
                 
                   Bacillus subtilis 
                 
                 Picossi et al. (2007) 
               
               
                 HupR 
                 Activator (global) 
                 
                   Vibrio vulnificus 
                 
                 Litwin &amp; Quackenbush (2001) 
               
               
                 HvrB 
                 Activator (global) 
                 
                   Rhodobacter 
                 
                 Buggy et al. (1994) 
               
               
                 IlvR 
                 Activator (local) 
                 
                   Caulobacter 
                 
                 Malakooti &amp; Ely (1994) 
               
               
                 IlvY 
                 Activator (local) 
                 
                   Escherichia coli 
                 
                 Wek &amp; Hatfield (1988) 
               
               
                 IrgB 
                 Activator (local) 
                 
                   Vibrio cholerae 
                 
                 Goldberg et al. (1991) 
               
               
                 LeuO 
                 Activator/Repressor 
                 
                   Salmonella enterica 
                 
                 Herna{acute over ( )}ndez-Lucas et al. (2008) 
               
               
                   
                 (global) 
                 
                   Typhimurium 
                 
               
               
                 LrhA 
                 Activator (global) 
                 
                   Escherichia coli 
                 
                 Lehnen et al. (2002) 
               
               
                 LysR 
                 Activator (local) 
                 
                   Escherichia coli 
                 
                 Stragier et al. (1983) 
               
               
                 MdcR 
                 Activator (local) 
                 
                   Klebsiella pneumoniae 
                 
                 Peng et al. (1999) 
               
               
                 MetR 
                 Activator (global) 
                   Streptococcus  spp. 
                 Kovaleva &amp; Gelfand (2007) 
               
               
                 MleR 
                 Activator (local) 
                 
                   Lactococcus lactis 
                 
                 Renault et al. (1989) 
               
               
                 MtaR 
                 Activator (global) 
                 Group B  streptococci   
                 Shelver et al. (2003) 
               
               
                 MvfR 
                 Activator (global) 
                 
                   Pseudomonas 
                 
                 Cao et al. (2001) 
               
               
                   
                   
                 
                   aeruainosa 
                 
               
               
                 NagR 
                 Activator (local) 
                 
                   Ralstonia eutropha 
                 
                 Jones et al. (2003) 
               
               
                 NahR 
                 Activator (local) 
                 NAH7 plasmid of 
                 Park et al. (2002) 
               
               
                 NhaR 
                 Activator (local) 
                 
                   Escherichia coli 
                 
                 Dover &amp; Padan (2001) 
               
               
                 NocR 
                 Activator (local) 
                 Ti plasmids of 
                 von Lintig et al. (1994) 
               
               
                   
                   
                 
                   Agrobacterium 
                 
               
               
                   
                   
                 
                   
                 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                 Specific 
                   
                   
                   
               
               
                 Super 
                 ligand 
                 Transcrip 
                 Operator 
                   
               
               
                 family 
                 activator 
                 tional 
                 sequence 
                 reference 
               
               
                   
               
             
            
               
                 AraC/ 
                 p- 
                 pobR 
                 GCCGGCGC 
                 http://www.pseudomonas.com/feature/ 
               
               
                 XylS 
                 hydroxy- 
                   
                 ATGCGCCG 
                 intergenic?start=280759&amp;stop= 
               
               
                   
                 benzoate 
                   
                 CCGGCCAG 
                 280935&amp;repliconid=136&amp;src=map 
               
               
                   
                   
                   
                 CCATAA 
                   
               
               
                   
               
               
                 LuxR 
                 N-(3-oxodo- 
                 LasR 
                 CTATGTCTT 
                 http://www.pseudomonas.com/feature/ 
               
               
                   
                 decanoyl) 
                   
                 TTGTTAG 
                 intergenic?start=1077905&amp;stop= 
               
               
                   
                 homoserine 
                   
                   
                 1078461&amp;repliconid=136&amp;src=map 
               
               
                   
                 lactone (3- 
                   
                   
                   
               
               
                   
                 oxo-C12- 
                   
                   
                   
               
               
                   
                 HSL) and N- 
                   
                   
                   
               
               
                   
                 butyryl 
                   
                   
                   
               
               
                   
                 homoserine 
                   
                   
                   
               
               
                   
                 lactone 
                   
                   
                   
               
               
                   
               
               
                 LTTR 
                 multiple 
                 mvfR 
                 TTCGGACTC 
                 http://www.pseudomonas.com/feature/ 
               
               
                   
                 quorum 
                   
                 CGAA 
                 intergenic?start=1077905&amp;stop= 
               
               
                   
                 sensing 
                   
                   
                 1078461&amp;repliconid=136&amp;src=map + 
               
               
                   
                   
                   
                   
                 Xiao et al 2006 
               
               
                   
               
               
                 LTTR 
                 flavonoids, 
                 nodD 
                 AGATTAGTA 
                 yang et al 2012; 
               
               
                   
                 naringenin, 
                   
                 AAATTGATT 
                 http://www.ncbi.nlm.nih.gov/gene/ 
               
               
                   
                 hesperetin 
                   
                 GTTGGGAT 
                 4403938 
               
               
                   
                   
                   
                 AGCTATCAT 
                   
               
               
                   
                   
                   
                 CCACGATAT 
                   
               
               
                   
                   
                   
                 GGATG 
                   
               
               
                   
               
               
                   
                 benzoate 
                 benR 
                 CCGAAAAA 
                 putativ, COWLESet al 2000, 
               
               
                   
                   
                   
                 GTACCGAA 
                 http://www.ncbi.nlm.nih.gov/gene/ 
               
               
                   
                   
                   
                 CATCCGTAA 
                 1046807 
               
               
                   
                   
                   
                 ATCTGGATA 
                   
               
               
                   
                   
                   
                 ACGTTCTGC 
                   
               
               
                   
                   
                   
                 ACAATCCG 
                   
               
               
                   
                   
                   
                 GATAGCCC 
                   
               
               
                   
                   
                   
                 CCCGCCAG 
                   
               
               
                   
                   
                   
                 CCGTCTCCC 
                   
               
               
                   
                   
                   
                 TAAC 
                   
               
               
                   
               
               
                 Lrp/ 
                 binding 
                 lysM 
                 TAAAATCGT 
                 Brinkman et al 
               
               
                 AsnC 
                 inhibition 
                   
                 ACCACTTAT 
                 2002+http://www.ncbi.nlm.nih.gov/ 
               
               
                   
                 with lysine 
                   
                 TACTAAAAA 
                 gene/1453332 
               
               
                   
                   
                   
                 CTTTTTCTA 
                   
               
               
                   
                   
                   
                 CACAAAACT 
                   
               
               
                   
                   
                   
                 AAGTTAGTA 
                   
               
               
                   
                   
                   
                 TCTAAC 
                   
               
               
                   
               
               
                 LTTR 
                 sulfur 
                 cysB 
                 TGTTGAAAT 
                 Hryniewicz et al, 1991; 
               
               
                   
                 sources? (no 
                   
                 TAAAGGCCT 
                 Delic-Atree 1997 + 
               
               
                   
                 cysteine) N- 
                   
                 TTAGAAACT 
                 http://www.pseudomonas.com/feature/ 
               
               
                   
                 acetyl-serine 
                   
                 TGAATTCTA 
                 show/?id=109899&amp;view=sequence 
               
               
                   
                   
                   
                 TGGACCGA 
                   
               
               
                   
                   
                   
                 ACTAAAA 
                   
               
               
                   
               
               
                 LTTR 
                 sulfur 
                 cysBH 
                   
                   
               
               
                   
                 sources? (no 
                   
                   
                   
               
               
                   
                 cysteine) N- 
                   
                   
                   
               
               
                   
                 acetyl-serine 
                   
                   
                   
               
               
                   
               
               
                 LTTR 
                 muconic acid 
                 benM 
                 See FIG. 8 
                   
               
               
                   
               
               
                 LTTR 
                 naringenin 
                 FdeR 
                 See FIG. 8 
                 Siedler S et al., 2014 
               
               
                   
               
               
                 LTTR 
                 salicylate 
                 NagR 
                   
                 Jones et al., 2003 
               
               
                   
               
               
                 LTTR 
                 salicylate 
                 NahR 
                   
                 Cebolla et al, 1997; van Sint Fiet et 
               
               
                   
                   
                   
                   
                 al, 2006; Calcagno review 
               
               
                   
               
               
                 LTTR 
                 protcatechuic 
                 PcaQ 
                 See FIG. 8 
                 MacLean et al, 2008 
               
               
                   
                 acid 
                   
                   
                   
               
               
                   
               
               
                 LTTR 
                 acetate 
                 AlsR 
                   
                 Fradrich et al, 2013, de Oliveira et 
               
               
                   
                   
                   
                   
                 al, 2013 
               
               
                   
               
               
                 LTTR 
                 L-arginin 
                 ArgP 
                 See FIG. 8 
                 Zhou et al, 2010; Laisram et al., 
               
               
                   
                   
                   
                   
                 1997 
               
               
                   
               
               
                 LTTR 
                 malonate 
                 MdcR 
                 See FIG. 8 
                   
               
               
                   
               
               
                 LTTR 
                   
                 AphB 
                 AACAACCTA 
                 Kovacikova et 2010, Bina et al 
               
               
                   
                   
                   
                 AGTTTGCA 
                 2015 
               
               
                   
               
               
                   
                 ROS, 
                 soxS 
                 TTTGCATAG 
                 Gil et al 
               
               
                   
                 superoxide, 
                   
                 CGTGAATAT 
                 2009+http://www.ncbi.nlm.nih.gov/ 
               
               
                   
                 paraquate 
                   
                 GTCAAAATT 
                 gene/1253251 
               
               
                   
                   
                   
                 GAT 
                   
               
               
                   
               
               
                 LTTR 
                 myricetin and 
                 kaeR 
                 
                   CGATTTGC 
                 
                 Pande et al 2001; 
               
               
                   
                 kaempferol 
                   
                 
                   CATTAATC 
                 
                 http://www.ncbi.nlm.nih.gov/gene/ 
               
               
                   
                   
                   
                 
                   CCATTAGG 
                 
                 ?term=kaeR 
               
               
                   
                   
                   
                 
                   ACTTTCGT 
                 
                   
               
               
                   
                   
                   
                   ATCGGA GA 
                   
               
               
                   
                   
                   
                 AGCCTTCAA 
                   
               
               
                   
                   
                   
                 CGTTATTAA 
                   
               
               
                   
                   
                   
                 ACATCATTG 
                   
               
               
                   
                   
                   
                 CTGGACCTT 
                   
               
               
                   
                   
                   
                 CTTGCGTCG 
                   
               
               
                   
                   
                   
                 GCCGTTTTA 
                   
               
               
                   
                   
                   
                 CCGTCCCTC 
                   
               
               
                   
                   
                   
                 CAGCACCAA 
                   
               
               
                   
                   
                   
                 TATAGCGGT 
                   
               
               
                   
                   
                   
                 AAACACCAG 
                   
               
               
                   
                   
                   
                 CCAATTCAG 
                   
               
               
                   
                   
                   
                 CATTTGGAT 
                   
               
               
                   
                   
                   
                 TCACAGCTA 
                   
               
               
                   
                   
                   
                 CGTTCGTCT 
                   
               
               
                   
                   
                   
                 CATGGTACT 
                   
               
               
                   
                   
                   
                 GGTTGGCA 
                   
               
               
                   
                   
                   
                 TGGGTTTTT 
                   
               
               
                   
                   
                   
                 AGCTCGGC 
                   
               
               
                   
                   
                   
                 CAATACTTT 
                   
               
               
                   
                   
                   
                 TCGTAAATC 
                   
               
               
                   
                   
                   
                 ATAAGGATC 
                   
               
               
                   
                   
                   
                 ATTTACCAT 
                   
               
               
                   
                   
                   
                 CAGATTACC 
                   
               
               
                   
                   
                   
                 TCCTATAAG 
                   
               
               
                   
                   
                   
                 TTGCTTACA 
                   
               
               
                   
                   
                   
                 ATCACCACT 
                   
               
               
                   
                   
                   
                 TTAAGGCAT 
                   
               
               
                   
                   
                   
                 AAAATCGTT 
                   
               
               
                   
                   
                   
                 GCAAACAAC 
                   
               
               
                   
                   
                   
                 TCAACTTTC 
                   
               
               
                   
                   
                   
                 GACTAA TG   
                   
               
               
                   
                   
                   
                 
                   TTATGCCT 
                 
                   
               
               
                   
                   
                   
                 
                   AAATGGAA 
                 
                   
               
               
                   
                   
                   
                 
                   TAATAAGA 
                 
                   
               
               
                   
                   
                   
                 
                   AGAAGGTT 
                 
                   
               
               
                   
                   
                   
                 
                   CTTCAAT 
                 
                   
               
               
                   
                   
                   
                 (5′)* 
                   
               
               
                   
               
               
                   
                 L-arabinose 
                 araC 
                 TCAGGCAG 
                 http://www.ecogene.org/gene/EG1 
               
               
                   
                   
                   
                 GATCCGCTA 
                 0054, 
               
               
                   
                   
                   
                 ATCTTATGG 
                 http://www.ncbi.nlm.nih.gov/gene/ 
               
               
                   
                   
                   
                 ACAAAAATG 
                 1251622 
               
               
                   
                   
                   
                 CTAATGCTT 
                   
               
               
                   
                   
                   
                 TGCAAAGT 
                   
               
               
                   
                   
                   
                 GTGACGCT 
                   
               
               
                   
                   
                   
                 GTGCAAATA 
                   
               
               
                   
                   
                   
                 TTCAATGTG 
                   
               
               
                   
                   
                   
                 GACATTCCA 
                   
               
               
                   
                   
                   
                 GCCATAGTT 
                   
               
               
                   
                   
                   
                 ATAGACACT 
                   
               
               
                   
                   
                   
                 TCTGTTACT 
                   
               
               
                   
                   
                   
                 TAATTTTAT 
                   
               
               
                   
                   
                   
                 CGCCTGAA 
                   
               
               
                   
                   
                   
                 CTGTACGCT 
                   
               
               
                   
                   
                   
                 TTTGTTACA 
                   
               
               
                   
                   
                   
                 AAGCGCTTT 
                   
               
               
                   
                   
                   
                 TCACAAGC 
                   
               
               
                   
                   
                   
                 GGGGTTGA 
                   
               
               
                   
                   
                   
                 TACGTGCTT 
                   
               
               
                   
                   
                   
                 TCATCAAGC 
                   
               
               
                   
                   
                   
                 GCAAAGTCT 
                   
               
               
                   
                   
                   
                 TGCGGAGA 
                   
               
               
                   
                   
                   
                 CGGAAGCT 
                   
               
               
                   
                   
                   
                 CTGTCGTCC 
                   
               
               
                   
                   
                   
                 TGGTCGATA 
                   
               
               
                   
                   
                   
                 TGGACAATT 
                   
               
               
                   
                   
                   
                 TGTTTC 
               
               
                   
               
               
                 *Bold sequences are DNA sites where KaeR bind. The site is palindromic meaning that it will bind to two somewhat complementary motifs (the two bold sequences). The sequence in between indicates space which is not necessarily needed to be matching this code in length nor sequence content. 
               
            
           
         
       
     
     Results 
     Onboarding a Prokaryote Transcription Activator to Yeast 
     To investigate the potential to build orthogonal biosensors using prokaryotic transcriptional activators in a eukaryotic chassis, we initially selected BenM from  Acinetobacter  sp ADP1 for several reasons. First, it belongs to the LTTR family, which is one of the most abundant families of transcriptional regulators found in a diverse range of prokaryotes. Second, in  Acinetobacter  sp ADP1, BenM serves as a native CCM-inducible transcriptional activator (Results,  FIG. 1 ). CCM is an intermediate from aromatic compound catabolism and an important precursor for bioplastics. Moreover CCM biosynthesis was recently refactored in yeast, yet without any high-throughput screening option available. Third, BenM has a well-characterized DNA binding site (herein termed BenO) and mode-of-action ( FIG. 1 a    and  FIG. 1 ). Finally, this protein does not require any binding to regulatory subunits apart from its cognate inducers, which should ensure its orthogonality in non-native chassis. 
     Engineering transcriptional repressors from prokaryotes into eukaryote chassis has emphasized the importance of operator positioning within synthetic eukaryote promoters in relation to transcriptional output. Hence, we first sought to identify optimal positioning of BenO when introduced into a eukaryote promoter. As a first expression cassette the full-length (491 bp) CYC1 promoter (CYC1p) was used to control the expression of green fluorescence protein (GFP) (Olesen, 3., Hahn, S. &amp; Guarente, L. Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner. Cell 51, 953-61 (1987)). CYC1p was recently reported as a suitable promoter for introduction of other non-native TF binding sites in yeast, and throughout this study all engineered reporter gene promoters will be based on chromosomally integrated full-length or truncated versions of this promoter. Initially, BenO was introduced into the 491 bp CYC1 promoter immediately upstream of one of the two TATA boxes—TATA-1β (designated 491 bp_CYC1p_BenO_T1) or TATA-2α (designated 491 bp_CYC1p_BenO_T2), or upstream of both (designated 491 bp_CYC1p_BenO_T1/T2)( FIG. 2 a   ). Outputs from these engineered promoters were compared by flow cytometry to expression from the native CYC1p (491 bp_CYC1p) using GFP as the reporter ( FIG. 1 a    and  FIG. 2 a   ). In general, introducing BenO negatively impacted the CYC1p activity ( FIG. 1 b   , white columns). However, when co-expressing BenM from the TEF1 promoter we observed 20-fold and 5-fold induction of expression from 491 bp_CYC1p_BenO_T1 and 491 bp_CYC1p_BenO_T1/T2 compared to the promoter activities without co-expression of BenM. For 491 bp_CYC1p_BenO_T2 we observed a modest 30% reduction in expression. Most importantly, BenM did not increase expression of native CYC1p without BenO ( FIG. 1 b   ). Taken together these data show that BenM can function as a transcriptional activator in yeast. 
     Protonated CCM is directly taken up by yeast at pH 4.5 without any growth defects ( FIG. 3 a - b   ). This enables CCM inducibility of the genetic devices to be tested by simple supplement of 200 mg/L CCM to the medium at pH 4.5. Following 24 h of cultivation GFP output was measured using flow cytometry. Here, we observed modest increases (1.3-2.2-fold,  FIG. 1 b   ) in reporter output from all CYC1 promoters that harbored BenO, whereas no change was observed from the native CYC1p ( FIG. 1 b   ). Also, all engineered promoters showed significant transcriptional activities in the control medium (no CCM) compared to background auto-fluorescence ( FIG. 1 b   ). 
     In order to lower the basal activity of the engineered promoters, we removed upstream activating sequences (UAS1 and UAS2) and introduced BenO into truncated versions of the CYC1p (designated 272 bp_CYC1p, 249 bp_CYC1p and 209 bp_CYC1p,  FIG. 2 a   ). Also, in order to improve the dynamic range of the genetic device we tuned the production of BenM by placing benM under the transcriptional control of three other native yeast promoters: TDH3p, RNR2p and REV1p. Together with TEF1p, this system allows for an expression range covering almost three orders of magnitude. By combining and chromosomally integrating all possible BenM expression cassettes with all CYC1p-derived reporter constructs, a total of 84 yeast strains were generated, including control strains ( FIG. 1 c   , Table 1 and  FIG. 4 ). Analyzing basal and CCM-induced GFP expression for all strains by flow cytometry we observed reporter outputs that spanned more than two orders of magnitude from the lowest to the highest GFP levels, with most of the high outputs resulting from reporters expressed from full-length CYC1p backbones co-expressed with BenM ( FIG. 1 c   ,  FIG. 4  and Table 2). Low-expressing strains mostly comprise truncated CYC1p reporter variants without BenO or BenM. These data showed that the BenO_T1 positioning allowed CCM-inducibility of all truncated variants of CYC1p, with the highest dynamic range observed for the minimal promoters 249 bp_CYC1p_BenO_T1 and 209 bp_CYC1p_BenO_T1 (3.2-4.7-fold)( FIG. 1 d    and Table 2). Among the genetic devices tested, strain MeLS0049 with 209 bp_CYC1p_BenO_T1 controlled by BenM expressed from REV1p showed both low basal activity and high CCM-inducibility (3.8-fold), and was therefore regarded as most suitable for application as a CCM biosensor. 
     High-Throughput Prototyping of Biosensors Variants 
     The dynamic range of a biosensor output is an important parameter when evaluating applicability of a biosensor for screening and selection. For this reason, we applied a high-throughput engineering strategy for identifying BenM mutants with higher dynamic ranges when expressed from the weak REV1 promoter. Previous mutagenesis studies identified residues important for ligand-binding in LTTR effector binding domains (EBDs). For this purpose we performed PCR-based mutagenesis of the BenM EBD (residues 90-304)( FIG. 2 a   ). Following mutagenesis we harnessed yeast&#39;s homologous recombination machinery for plasmid gap repair of variant EBDs with the BenM DNA-binding domain (DBD)( FIG. 2 a   ). A population derived from approx. 40,000 transformants was analyzed by fluorescence activated cell sorting (FACS) using a two-step approach, in which we first removed the variants showing increased basal activity. Next, we compared fluorescence output from the population of transformants in control and CCM medium ( FIG. 2 b   ). From this, all cells showing higher fluorescence than the fluorescence observed in control medium were sorted ( FIG. 2 b   ). Sorted cells were subsequently cultivated as clones and validated by flow cytometry ( FIG. 2 c   ). 
     Here we identified five BenM variants with higher dynamic ranges than wild-type BenM ( FIG. 2 c   ). Sequencing of the BenM variants identified a triple mutant with point mutations H110R, F211V and Y286N in the BenM EBD ( FIG. 2 c   ). Plasmid-based expression of BenM H110R, F211V, Y286N  showed doubled GFP output upon CCM induction (6-fold), compared to induction for the plasmid-based expression of wild-type BenM ( FIG. 2 c   ). Interestingly, the mutations in BenM H110R, F211V, Y286N  were not positioned in the immediate vicinity of the CCM binding site ( FIG. 2 d   ). Similar to all other genetic devices engineered in this study, BenM H110R, F211V, Y286N  was also integrated into the genome for stable expression. 
     LTTR-Based Biosensor Specificity and Orthogonality 
     To assess the potential application of the LTTR-based biosensor for CCM in yeast, we next investigated the specificity of BenM, as well as its potential impact on the host transcriptome. First, by testing a range of diacids supplied to the growth medium at pH 4.5 with identical molar concentrations to CCM (1.4 mM), we observed that among the diacids tested both BenM and BenM H110R, F211V, Y286N  induce GFP expression specifically in response to CCM ( FIG. 3 a   ). Second, to test for transcriptional orthogonality of BenM H110R, F211V, Y286N  in yeast, we used RNA-seq to quantify and compare the transcriptomes of cells with (MeLS0284) or without (MeLS0138) expression of BenM H110R, F211V, Y286N . As the genetic device has low basal activity ( FIG. 4  and Table 2) we analyzed yeast transcriptomes following 24 h cultivation in the presence of CCM. Here, we observed that the average GFP transcript abundance from strain MeLS0284 was approximately 27-fold higher compared to strain MeLS0138 ( FIG. 3 b   ,  FIG. 5 ). Apart from genes encoding GFP and BenM, only one other gene encoding the Golgi-associated retrograde protein complex component TCS3, passed our stringent cut-off (P&lt;0.05, &gt;2-fold) showing a modest decrease (2.3×) in expression level when BenM H110R, F211V, Y286N  was expressed ( FIG. 3 b   ). We found no match to BenO in this gene&#39;s promoter (data not shown), suggesting that the minor transcriptome perturbations could be due to noise in RNA-seq measurements or indirect effects. 
     A Design for Onboarding LTTR-Based Biosensors in Yeast 
     The genetic device developed in this study represents to the best of our knowledge the first example of transplanting a prokaryotic transcriptional activator into a eukaryotic chassis and successfully using it to activate gene expression without the need for modifying the protein beyond codon optimization. Acknowledging the vast numbers of transcriptional activators found among LTTR members, the optimal reporter promoter design (209 bp_CYC1p_BenO_T1) could prove valid for other metabolic engineering and biotechnological applications. To test the generality of the biosensor design for onboarding other small-molecule binding transcriptional activators as biosensors in yeast we selected four other candidates from the LTTR family; FdeR from  Herbaspirillum seropedicae , PcaQ from  Sinorhizobium meliloti , ArgP from  Escherichia coli , and MdcR from  Klebsiella pneumonia , with co-inducers naringenin, protocatechuic acid (PCA), L-arginine, and malonic acid, respectively. In this proof-of-principle study we selected the four candidates based on a minimal set of information, including knowledge about operator sequences, experimental evidence for ligand-inducible control of target operons, and their mode-of-action within native chassis (ie. activation,  FIG. 1 ). Furthermore all of these metabolites can passively diffuse across the yeast plasma membrane, with the exception of malonic acid, which requires the expression of the dicarboxylic acid transporter MAE1 from  Schizosaccharomyces pombe . For this purpose, the gene encoding MAE1 was integrated into cells expressing MdcR (Table 3). Based on this knowledge, and the aforementioned selection criteria, we directly replaced BenO located in the T1 position of the 209 bp_CYC1p promoter with operator sequences for each of these LTTRs ( FIG. 4 a   ,  FIG. 2 a - b   , Table 4). We first tested if expression of GFP could be activated upon low and high expression of individual LTTRs. From this, it was evident that all LTTRs were able to activate GFP expression from the 209 bp_CYC1p_T1 promoter when the LTTR was expressed from the strong TDH3 promoter compared to yeast cells without expression of an LTTR (1.4×-8.1×), with BenM showing the strongest activation (8.1×)( FIG. 4 a   ). Similarly, GFP expression could also be induced by ArgP when the weak REV1p promoter controlled expression of the LTTR (2.2×). This proves the broad applicability of the reporter promoter design, and that biosensor output is tunable depending on the expression level of the LTTR. Next, we tested if each LTTR could further induce GFP expression when its cognate inducer was supplied to the growth medium ( FIG. 4 b   ). For this purpose we prepared medium with either 1.4 mM CCM, 0.2 mM naringenin, 30 mM L-arginine, 1.4 mM PCA, or 10 mM malonic acid, as previously reported to be relevant concentrations in terms of bio-based production and microbial physiology. Here, in addition to BenM, ArgP was the only LTTR enabling a significant ligand-inducible increase in GFP expression when LTTR expression was controlled by REV1p ( FIG. 4 b   ). However, when expressing LTTRs from the TDH3 promoter all LTTRs, except PcaQ, significantly increased GFP expression (1.4×-4.1×) when their cognate ligand was present in the cultivation medium ( FIG. 4 b   ). Taken together all tested LTTRs were able to activate expression of GFP when their operators were placed in the T1 position of the 209 bp_CYC1p scaffold promoter (Table 4). Furthermore, just as for BenM, yeast expressing FdeR, ArgP and MdcR from the strong TDH3 promoter, were able to further induce GFP expression upon addition of their cognate inducers ( FIG. 4 b   ). 
     Many of the characterized LTTRs regulate operons by binding prototypic LTTR box patterns 5′-T-N11-A-3′ and 5′-TTA-N7/8-GAA-3′. In addition to transcriptional orthogonality ( FIG. 3 b   ), we therefore further tested if individual LTTRs would cross-react with operators for another LTTR. For this purpose, we expressed LTTRs ArgP and MdcR together with the 209 bp_CYC1p_T1 promoter with operators for MdcR (herein MdcO) or ArgP (herein ArgO) driving the expression of GFP. As controls we tested GFP expression from 209 bp_CYC1p_T1 promoter with MdcO or ArgO without expression of LTTRs. Flow cytometry analysis showed specificity between LTTR transcriptional activators and their inferred operator ( FIG. 4 c   ). This is in agreement with another study on cross-reactivity between promoter and transcriptional regulators of the TetR family, and the fact that LTTR residues in both the conserved N-terminal DNA-binding domains and the divergent EBDs are important for DNA-binding. 
     In Vivo Application of LTTR-Based Biosensors in Yeast 
     Based on our engineering efforts and characterization of prokaryote LTTR-based biosensors imported into yeast, we next addressed whether such biosensors would support real-time monitoring of product accumulation in vivo and thereby potentially provide high-throughput screening assays of biocatalysts. To test this we selected CCM and naringenin, for which highest titers in shake-flask cultivated haploid yeast of approx. 1 mM (141 mg/L) and 0.2 mM (54 mg/L), respectively, have recently been reported. Also, these two products are of general interest to biotechnology with CCM being a platform chemical for the production of several valuable consumer bio-plastics, whereas naringenin belongs to a class of secondary metabolites called flavonoids with nutritional and agricultural value. 
     Before applying the biosensors for in vivo detection of these metabolites we first tested their operational range and induction kinetics. For BenM and BenM H110R, F211V, Y286N , we observed a weakly sigmoidal input-output relationship between CCM concentration and GFP output following 24 h cultivation. For chromosomally integrated BenM H110R, F211V, Y286N  and BenM, a maximum of 10- and 3.5-fold induction was reached in the presence of the highest soluble CCM concentrations (1.4 mM, 200 mg/L)( FIG. 5 a   ). Interestingly, induction kinetics of BenM and BenM H110R, F211V, Y286N  were similar. This is in line with BenM mutations likely not to be involved with direct binding of CCM ( FIG. 2 d   ), but rather alter BenM binding to DNA to support increased GFP expression. 
     Similarly, for FdeR we first tested naringenin sensitivity and operational range of the sensor. As for CCM, the operational range was only tested for concentrations of naringenin soluble in growth medium (ie. &lt;0.2 mM). Here, we observed that expression of FdeR controlled by the weak REV1 promoter did not support induction of GFP expression at any of the tested concentrations ( FIG. 5 b   ), yet when expression of FdeR was controlled by the strong TDH3 promoter a maximum 1.7-fold increase in GFP expression was observed following 24 h cultivation in the presence of 0.2 mM naringenin ( FIG. 5 b   ). Taken together, the operational ranges of BenM and FdeR are within the ranges of reported CCM and naringenin production titers in yeast, and therefore could make them applicable for screening such biocatalysts. 
     Next, we transformed the CCM biosensor (209 bp_CYC1p_BenO_T1::GFP and REV1p::BenM H110R, F211V, Y286N ) into a small library of six yeast strains engineered to produce CCM. CCM production with a final titer of 149 mg/L was recently reported in haploid yeast using a three-step heterologous pathway consisting of a AroZ homologue from  Podospora anserina  encoding dehydroshikimate dehydratase (PaAroZ), the AroY gene from  Klebsiella pneumonia  encoding the multi-subunit protocatechuic acid decarboxylase (PCA-DC) and the CatA gene encoding catechol 1,2-dioxygenase from  Candida albicans  (CaCatA) ( FIG. 6 a   ). From that study it was clear that PCA-DC was a rate-limiting step for flux through the upper part of the shikimate pathway towards CCM. It was also suggested that an increased supply of precursor towards erythrose-4-phosphate (E4P) could improve CCM production. For this reason we introduced single or multiple copies of different PCA-DC subunits from  K. pneumonia  and introduced no or one additional copy of transketolase (Tkl1) from  S. cerevisiae  ( FIG. 6 a   ). The six-membered CCM production strain library and a wild-type CCM null background strain were cultured individually. After 24 h of cultivation the medium was analyzed for CCM concentration using HPLC and the cells were analyzed by flow cytometry for GFP intensity measurements. Here, we observed a strong correlation (r=0.98) between GFP output and CCM production titers, spanning a range of 0.00016-1.39 mM (0.023-197.6 mg/L)( FIG. 6 b   ). The highest titers were obtained in strain ST4245-2 with multiple TY integrations of AroY subunits B and C and Tkl1 ( FIG. 6 a - b   ). To further examine the performance of the CCM biosensor we monitored GFP output and CCM production titers following 72 h of cultivation. Here, GFP outputs were saturated at titers &gt;1.41 mM (200 mg/L)( FIG. 6 a - b   ). However, the strain that produced the most CCM after 72 h (3.03 mM, 430.8 mg/L) also produced the most CCM and had the highest fluorescence after 24 h, emphasizing the applicability of the CCM biosensor for screening high-producing strains during early stages of cultivation. 
     Finally, we transformed 209 bp_CYC1p_FdeO_T1::GFP and TDH3p::FdeR into yeast strains with a 5-step heterologous naringenin pathway. For building a small library of naringenin producing strains, we chromosomally introduced either in single copy of the pathway (EVR1), or with one and two additional integrations of bottleneck enzymes (AtPAL-2 and HaCHS for EVR2; AtPAL-2, HaCHS, and AtC4H:L5:AtATR2 for EVR3)( FIG. 6 c   , Table 1). Following 48 h of cultivation the medium was analyzed for naringenin concentration using UPLC and the cells were analyzed by flow cytometry for GFP intensity measurements. As observed for the CCM biosensor, the naringenin biosensor also had a strong correlation (r=0.96) between GFP output and naringenin titers, spanning a range of 0.094-0.184 mM (25.61-50.18 mg/L)(FIG.  6   d ), with the highest titer obtained in strain EVR3 containing two additional integrations of bottleneck enzymes on top of the full copy of the 5-step naringenin pathway. For the naringenin sensor we observed a poorer correlation between biosensor output and titers at 24 h (r=0.87) compared to our 48 h (r=0.96) measurements ( FIG. 6 c - d   ). However, just as for the CCM biosensor, the strain that produced the most naringenin at 48 h (0.184 mM, 50.18 mg/L) also produced the most naringenin (0.045 mM, 12.25 mg/L) and had the highest fluorescence at 24 h. 
     Taken together, the two applications of the LTTR-based biosensors suggest that simple expression of the LTTR and an engineered reporter promoter (209 bp_CYC1p_T1::GFP) with an operator site in position T1 allows for direct transplantation of prokaryotic transcriptional activators as biosensors to screen for the best-performing biocatalysts. Interestingly, though some of the transcriptional activators used in this study derived from prokaryotes with growth optima at higher temperatures compared to yeast, BenM showed a higher dynamic range in output at 30° C. compared to 37° C. ( FIG. 7 ), illustrating robustness of LTTR performance. 
     Application of Biosensors in CHO Cells 
     For testing the reporter promoter design with other promoter backbones AND in another eukaryotes, Chinese hamster ovary cells was transformed using the human cytomegalovirus promoter backbone (CMV) instead of the CYC1 promoter backbone used in yeast. 
     Just as in the case with the yeast design using CYC1 promoter as a backbone, the present inventors put the binding site (benO) for the prokaryotic transcriptional activator BenM 6 bp upstream of the TATA box the CMV promoter and scored reporter gene activity (GFP fluorescence) in the presence and absence of the transcriptional activator BenM. As can be seen in  FIG. 13 , the design worked when putting into another promoter backbone AND another host organism (CHO cells). 
     In addition to this, the present inventors also tested 17 different positions for positioning of the BenM binding site (benO). Only position 6 upstream of the TATA box gave a significant response (See  FIG. 14 ). 
     Prokaryotic operator benO for the prokaryotic transcriptional activator BenM is placed 6 bp upstream of the TATA box the CMV promoter 
     
       
         
           
               
               
               
            
               
                   1 
                 GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA 
                   
               
               
                   
               
               
                  51 
                 TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA 
               
               
                   
               
               
                 101 
                 TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA 
               
               
                   
               
               
                 151 
                 TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA 
               
               
                   
               
               
                 201 
                 TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA 
               
               
                   
               
               
                 251 
                 TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG 
               
               
                   
               
               
                 301 
                 CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC TACTTGGCAG 
               
               
                   
               
               
                 351 
                 TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA 
               
               
                   
               
               
                 401 
                 GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC 
               
               
                   
               
               
                 451 
                 TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG 
               
               
                   
               
               
                 501 
                 GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG 
               
               
                   
               
               
                 551 
                 TAGGCGTGTA CGGT GGATAC TCCATAGGTA TTTTATTATA CAAATAATGT   
               
               
                   
               
               
                 601 
                   GTTTGAACTT ATTAAAACAT TCTTTTAAGG TATAAACAA G AGGTC  T 
               
               
                   
               
               
                 651 
                 AAGCAGAGCT  C     
               
               
                   BenO  (Bold) 
               
               
                     (Bold, Italic) 
               
               
                     (BoldfItalic, underlined) 
               
               
                   START CODON  (underlined) 
               
            
           
         
       
     
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