Yeast pheromone pathway

Schematic of induction and repression of a-specific, alpha-specific, and haploid-specific genes in different mating types

Yeast Saccharomyces cerevisiae exist either in haploid or diploid state. The two mating types are called MATa and MATx and differ only within approx. 2 kbp long region on chromosome III called MAT locus. MATa locus expresses transcription factors a1 and a2, whereas MATx locus expresses transcription factors α1 and α2. Both types express three groups of genes, which are:

• haploid-specific genes (h-sg)
• a-specific genes (a-sg)
• α-specific genes (α-sg)

As their names indicate, these genes are only active in haploids, MATa cells, or MATx cells, respectively.

Both mating types continuously produce small amounts of mating type-specific pheromone and when the two cells of opposite types are in close proximity, they identify each other by sensing each other’s pheromone. The response to detected pheromone is facilitated by so-called pheromone pathway, which is a cascade of chemical reactions that results in the preparation for mating. Since only haploids mate, the yeast pheromone pathway is only functional in haploid cells.

Design

Reconstruction of yeast pheromone pathway in diploid cell

Schematic of the wild-type yeast pheromone pathway.

The schematic shows that h-sg genes are switched off in a diploid cell and since all genes playing a role in the yeast pheromone pathway signal transduction (except for the pheromone receptor) are h-sg, the pathway can be reconstructed by switching these genes on. It can be managed by interrupting one of the h-sg regulators - a1 or α2. Since a1 has no function in a haploid, its deletion has no effect in a haploid cell, but it interrupts the repression of h-sg in a diploid.

Repression and induction schematic of the synthetic MATa/α strains

After a wild-type haploid is exposed to a mating pheromone, the yeast pheromone pathway induces the expression of mating genes that initiate the mating process. Since it is not desired for the synthetic diploid to mate, the initiation of the mating process needs to be disrupted. It can be achieved by repressing transcription of Ste12 in the diploid. However, since Ste12 is fundamental in both haploid types for the process of mating, the designed mechanism must preserve transcription in haploid cells and repress it in a diploid.

For this purpose, mechanism of tetracycline-controlled transcriptional activation was used: a1 gene in MATa was replaced by TetR and STE12 was put under the control of a synthetic a-specific promoter that is repressed in the same way as a-sg. In MATx, α1 repression was preserved by placing it under the control of pTet and STE12 was also put under the control of pTet. As a result, transcription of Ste12 is preserved in both haploids and repressed in a diploid - pTet-STE12 thanks to TetR repressor that is expressed from MATa chromosome, and a-specific STE12 thanks to natural repression of a-sg in diploid. What is the most important, h-sg are expressed in the diploid as a result of the a1 absence, thus preserving transcription of all the genes that the yeast pheromone pathway consists of.

There is no wild-type Ste12 in the synthetic diploid, but some pheromone-depemdent transcriptional activation needs to be preserved in order to induce synthetic genes. For this purpose, synthetic Ste12 is used whose DNA-binding domain, that binds to specific sequence within the promoters of mating genes, was replaced by Gal4p DNA-binding domain. This part of Gal4 transcription factor binds to Gal1 promoter that can further turn on transcription of any following gene, in this case synthetic pheromone or location tag. As a result synthetic Ste12 does not induce transcription of mating genes but preserves the ability to be activated by the reconstructed pheromone pathway and further induce transcription of synthetic genes.

MATx integration plasmid

MATx integration plasmid carries a synthetic MATx locus that integrates into the wild-type locus putting both α1 and α2 transcription factors on synthetic promoters and also inserting STE12 gene with its promoter into the locus.

Wild-type MATx locus

Wild-type MATx locus carries genes that code for α1 and α2 transcription factors. The locus codes for α1 in one direction and for α2 in the opposite direction simultaneously. Therefore any changes in α1 gene’s promoter would disrupt the promoter of α2. This problem was resolved by putting also the α2 on a synthetic promoter.

Integrated parts

All parts are cloned into the pRSII406 integrating vector (from Addgene). The final plasmid shown in the picture includes a PstI restriction site used for linearization prior to chromosomal integration. The whole plasmid is integrated between the α1 and α2 ORFs.

α1 on pTet promoter

ORF of α1 is a genomic sequence, it also serves as a homologous part for the plasmid integration. pTet promoter has sequence taken from ^[Ellis2009] (the T16 version). This promoter is repressed by TetR (tetracycline) and is active in the absence of TetR (see this Wikipedia page for more information about tetracycline-controlled transcriptional activation).

See part sequence

α2 on CYC1 promoter

ORF of α2 is a genomic sequence, it also serves as the second homologous part for the plasmid integration. CYC1 promoter has sequence taken from ^[Curran2014] (the CYC1v3 version).

See part sequence

For both α1 and α2, no synthetic terminators were designed because the terminators (native) will be already within the chromosome after integration.

STE12 on pTet promoter

ORF of STE12 is a genomic sequence that was extracted from chromosomal DNA also with the 3’UTR. Therefore, no synthetic terminator was included. Promoter pTet has sequence taken from ^[Ellis2009].

See part sequence

The MATx plasmid

MATa integration plasmid

MATa integration plasmid carries a synthetic MATa locus that integrates into the wild-type locus inserting TetR and STE12 with their promoters into the center of the a1 ORF and thus disrupting it.

Integrated parts

All parts are cloned into the pRSII406 integrating vector (from Addgene). The final plasmid shown in the picture includes a SnabI restriction site used for linearization prior to chromosomal integration. The whole plasmid is integrated within the a1 ORF.

TetR on ADH1 promoter

Tetracycline repressor ORF sequence was taken from Addgene plasmid sequence, the ADH1 promoter sequence was also taken from Addgene plasmid sequence. CYC1 terminator is included after TetR, it’s sequence was also taken from Addgene plasmid sequence.

See part sequence

STE12 on a-specific CYC1 promoter

ORF of STE12 is a genomic sequence that was extracted from chromosomal DNA also with the 3’UTR. Sequence of a-specific CYC1 promoter was obtained from Prof. Vershon, who presented series of a-specific promoters ^[Zhong1999]. It is a CYC1 promoter with inserted α2-Mcm1 binding sequence from AGA2 gene (α2-Mcm1 complex represses a-sg).

See part sequence

Parts of a1 ORF

Two parts of a1 OFR are included as homologous parts for chromosomal integration. The plasmid integrates into the center of a1 ORF and disrupts the gene.

See part sequence

The MATa plasmid

Synthetic Ste12

Authors of ^[Pi1997] constructed over 34 hybrid proteins combining domains of two different transcriptional factors. We obtained the synthetic Ste12 transcription factor from the authors of the paper. It consists of STE12 transcriptional activation domain and GAL4 DNA-binding domain, and is expressed from ADH1 promoter. GAL4 is required for activating GAL genes in the absence of glucose.

As the schematic shows, the site for repression by Gal80p is missing in this synthetic transcription factor, as a result this hybrid system works also in presence of glucose. The STE12 activation domain has the sites for repression by Dig1p and Dig2p, which are release from this site only in presence of pheromone.

Construction

Construction of reporter plasmids

The pADH1, pSTE2, pSTE5, and pFUS1 promoters were obtained by PCR from yeast genome (isolated according to standard protocol from 7283 MATx strain). The asCYC1 and pTv3 promoters were obtained by PCR from g-blocks. The primers used for this are as follows:

All promoters were amplified in a single PCR run, with the following conditions:

PCR products were then gel verified

INSERT PHOTO_1

and were then purified (insert purification kit name) and restricted by corresponding restriction enzymes, which are listed in the following table

The corresponding vector for reporter plasmids was prepared by restriction and ligation of yeGFP and CYC1 terminator into a pRSII416 CEN plasmid (obtained from Addgene).

Construction of INSERT MATa and INSERT MATx

The sequence of INSERT MATa was ordered as a single gBlock yG_MATa with restriction sites at the ends. The sequence of INSERT MATx was ordered as two gBlocks, yG_MATx_1 and yG_MATx_2. Upon arrival, the gBlocks were resuspended in TE buffer according to the protocol of the supplier (IDT). All gBlocks were to be cloned to the backbone pRSII415 (obtained from Addgene). To achieve this, the gBlocks were restricted by the following enzymes, along with the vector

The restrictions were all 50 ul reactions with 500 ng of DNA run with standard protocol. Enzymes and the restriction buffer were obtained from NEB. Restrictions were purified (insert purification kit name) and ligated together overnight for 16 hours at 16°C. Ligations were then inactivated in 65°C for 15 mins. Inactivated ligations were transformed to EC E5alpha cells. After 30 min incubation in 37°C, 400 ul of each transformation was plated on a separate LB-M plate supplied with Ampicillin resistance. Plates were left overnight in 37°C. The following day, patches were streaked from four colonies from the INSERT MATa plate, and from four colonies from the INSERT MATx plate. Patches were plated on separate LB-M Ampicillin plates for INSERT MATa and INSERT MATx respectively. Plates with patches were left overnight in 37°C. The following day, plasmid DNA was obtained from the patches using a miniprep kit(). Obtained plasmids were gel verified (see Validation).

Results

Final constructs

Verification of constructed plasmids

Integration plasmids

Plasmids of INSERT MATa and INSERT MATx were restricted by NEB enzymes EcoRI and HindIII (MATa) and SpeI and HindIII (MATx) in NEB CutSmart Buffer for 60 min in 37°C. Restrictions were loaded in an NaOH gel. The gel was run for 90 mins at 90V.

Reporter plasmids

Validation of the constructs in yeast

Mating test of synthetic haploids

Reporter plasmids' characterisation

Materials and methods

Used strains

1. Ecoli E5alpha
2. 7283 MATx yeast strain
3. 7284 MATa yeast strain
4. 10150 MATa/x yeast strain
5. 6193 MATa yeast strain (URA LEU HIS)
6. 6194 MATx yeast strain (URA LEU TRP)

Used material

1. LB-M agar plates with chloramphenicol
2. LB-M agar plates with ampicillin
3. 1.5 ml eppendorf tubes
4. 0.5 ml PCR tubes
5. 50 ml centrifuge conical base and rim tubes
6. NucleoSpin Plasmid DNA, RNA, and protein purification Kit
7. NucleoSpin Gel and PCR Clean-up Kit
8. LB-M medium with chloramphenicol
9. NaOH agarose gel and buffer
10. Sphero Rainbow Calibration Particles, 8 Peaks, 3.0-3.4

Used methods

1. Transformation
2. Miniprep
3. Restriction digest
4. Ligation
5. NucleoSpin Gel Clean-up
6. NucleoSpin Plasmid DNA purification
7. Flow cytometry

All used protocols can be found here: Protocols

Used software

1. CFlow Plus
2. Microsoft Excel
3. Sphero PMT QC Template

Used Parts

INSERT MATa :

AATTCATCTAGAGAAGAAAGCAAAGCCTTAATTCCAAGGAAAAAGAAGAAGTTGCAAAGAAATGTGGCATTACTCCACTTCAAGTAAGAGTTTGGGTATGTAATATGAGAATCAAACTTAAATATATCCTATACGTAGTATGGCGGAAAACATAAACAGAACTCTGTTTAACATTCTAGGTACTGAGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATCTAAAATTCCCGGGATCCGCTGTACGCGGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTGCGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATTTTAATAAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGCAGAAAAAGAAACAAGGAAGAAAGGGAACGAGAACAATGACGAGGAAACAAAAGATTAATAATTGCAGGTCTATTTATACTTGATAGCAAGACAGCAAACTTTTTTTTATTTCAAATTCAAGTAACTGGAAGGAAGGCCGTATACCGTTGCTCATTAGAGAGTAGTGTGCGTGAATGAAGGAAGGAAAAAGTTTCGTGTGCTTCGAGATACCCCTCATCAGCTCTGGAACAACGACATCTGTTGGTGCTGTCTTTGTCGTTAATTTTTTCCTTTAGTGTCTTCCATCATTTTTTTGTCATTGCGGATATGGTGAGACAACAACGGGGGAGAGAGAAAAGAAAAAAAAAGAAAAGAAGTTGTAAACCCACACCGGGTGTCATAATCAACCAATCGTAACTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAACAAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAGGGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCTTCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGTCCACTAGTCCCGGGAGCAAGATCAAGATGTTTTCACCGATCTTTCCGGTCTCTTTGGCCGGGGTTTACGGACGATGGCAGAAGACCAAAGCGCCAGTTCATTTGGCGAGCGTTGGTTGGTGGATCAAGCCCACGCGTAGGCAATCCTCGCAGATCTCGAACCATGTAATTTCCGAATACGGTAATTACACGCATCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCCTTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACACACAAACACAAATACACACACTAAAAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATCGACCGGTCGAGGAGAACTTCTAGTATATCTACATACCTAATATTATTGCCTTATTAAAAATGGAATCCCAACAATTACATCAAAATCCACATTCTCTTCAAAATCAATTGTCCTGTACTTCCTTGTTCATGTGTGTTCAAAAACGTTATATTTATAGGATAATTATACTCTATTTCTCAACAAGTAATTGGTTGTTTGGCCGAGCGGTCTAAGGCGCCTGATTCAAGAAATATCTTGACCGCAGTTAACTGTGGGAATACTCAGGTATCGTAAGATGCAAGAGTTCGAATCTCTTAGCAACCATTATTTTTTTCCTCAACATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGATGACTGGAAATTTTTTGTTAATTTCAGAGGTCGCCTGACGCATATACCTTTTTCAACTGAAAAATTGGGAGAAAAAGGAAAGGTGAGAGCGCCGGAACCGGCTTTTCATATAGAATAGAGAAGCGTTCATGACTAAATGCTTGCATCACAATACTTGAAGTTGACAATATTATTTAAGGACCTATTGTTTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATAGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGCTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACAGGTAGTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAATTTCCTACATTGAAGGTTCAACATCAATTGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCAACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTTGGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATCTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATTGAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAGTACCACCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAAAAGATTCTCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATAATAGAAACGACACGAAATTACAAAATGGAATATGTTCATAGGGTAGACGAAACTATATACGCAATCTACATACATTTATCAAGAAGGAGAAAAAGGAGGATGTAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAACGTGATAAGGAAAAAGAATTGCACTTTAACATTAATATTGACAAGGAGGAGGGCACCACACAAAAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATATTGGAGGATTTTCTCTAAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCATTTGATGGAGTTTAAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAATTTTACTCTGTCAGAAACGGCCTTAACGACGTAGTCGATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGG

INSERT MATx :

ATACTTTTACCGGTATTTTGTCTGTAATTTATTCTCTATCACTGATAGGGACTTCTCTATCACTGATAGGGAACCCAGCCTGATTTATACTATTAGGGATCGCAGGAAGGCGGTGGGAAGTCCGGGAGTCGCTGAGGGGAAGTGTCAGTGGTTTTGGGTATAAATGGCTGGTTGTTCCCTATCAGTAATAGAGAATTCCCTATCAGTGATAGAGACTGCGGATTTAGAAACTACCTGATAAAAGTATCAACAAAAATTGCGCATGCCGGCCTGGATTTTGCGCAAATTTACCTTAACGTCCCACAATATGTTTACTTCGAAGCCTGCTTTCAAAATTAAGAACAAAGCATCCAAATCATACAGAAACACAGCGGTTTCAAAAAAGCTGAAAGAAAAACGTCTAGCTGAGCATGTGAGGCCAAGCTGCTTCAATATTATTCGACCACTCAAGAAAGATATCCAGATTCCTGTTCCTTCCTCTCGATTTTTAAATAAAATCCAAATTCACAGGATAGCGTCTGGAAGTCAAAATACTCAGTTTCGACAGTTCAATAAGACATCTATAAAATCTTCAAAGAAATATTTAAACTCATTTATGGCTTTTAGAGCATATTACTCACAGTTTGGCTCCGGTGTAAAACAAAATGTCTTGTCTTCTCTGCTCGCTGAAGAATGGCACGCGGACAAAATGCAGCACGGAATATGGGACTACTTCGCGCAACAGTATAATTTTATAAACCCTGGTTTTGGTTTTGTAGAGTGGTTGACGAATAATTATGCTGAAGTACGTGGTGACGGATATTGGGAAGATGTGTTTGTACATTTGGCCTTATAGAGTGTGGTCGTGGCGGAGGTTGTTTATCTTTCGAGTACTGAATGTTGTCAGTATAGCTATCCTATTTGAAACTCCCCATCGTCTTGCTGCAGAGTAGTGTCTGAGGTACAAACATCTTAGTAGTGTCGAGAGGGTTGATTGTTTATGTATTTTTGCGAAATATATATATATATATTCTACACAGATATATACATATTTGTTTTTCGGGCTCATTCTTTCTTCTTTGCCAGAGGCTCACCGCTCAAGAGGTCCGCTAATTCTGGAGCGATTGTTATTGTTTTTTCTTTTCTTCTTCTATTCGAAACCCAGTTTTTGATTTGAATGCGAGATAAACTGGTATTCTTCATTAGATTCTCTAGGCCCTTGGTATCTAGATATGGGTTCTCGATGTTCTTTGCAAACCAACTTTCTAGTATTCGGACATTTTCTTTTGTAAACCGGTGTCCTCTGTAAGGTTTAGTACTTTTGTTTATCATATCTTGAGTTACCACATTAAATACCAACCCATCCGCCGATTTATTTTTCTGTGTAAGTTGATAATTACTTCTATCGTTTTCTATGCTGCGCATTTCTTTGAGTAATACAGTAATGGTAGTAGTGAGTTGAGATGTTGTTTGCAACAACTTCTTCTCCTCATCACTAATCTTACGGTTTTTGTTGGCCCTAGATAAGAATCCTAATATATCCCTTAATTCAACTTCTTCTTCTGTTGTTACACTCTCTGGTAACTTAGGTAAATTACAGCAAATAGAAAAGAGCTTTTTATTTATGTCTAGTATGCTGGATTTAAACTCATCTGTGATTTGTGGATTTAAAAGGTCTTTAATGGGTATTTTATTCATTTTTTCTTAGTGTGTGTATTTGTATTTGCGTGTCTATAGAAGTATAGTAATTTATGCTGCAAAGGTCCTAATGTATAAGGAAAAAAAATTTAGAGAAAAAAAGAAAAAAAGAGTTTTATATACATACAGAGCACATACATGCCATATAATCATGTATATACGCGCACATATATATATGCCTGTATGTGTCAGCACTAAATTTACCTGAACATACGCGCTATATATACGCGCCTCGCGTATATGCTCGAGGATTCCCTACGCGTGGGCTTTTTTTACTAACCAACGCGCGCGAAATACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCGACTACGTCGTTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCATTATGGGAAATGGTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCATTGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATATAAATTAGGAATCATAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTCCTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATTAGTATCAATTTGCTTACCTGTATTCCTTTACATCCTCCTTTTTCTCCTTCTTGATAAATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTTGACCTTATTCTTTGGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGTAGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGGCCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAACACTACCTGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAGCCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGAACACCTGTAGCATCTATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATTAGAATGGTATATCCTTGAAATATATATATATATATTGCTGAAATGTAAAAGGTAAGAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCTTCTCTATTCTATATGAAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGTATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCTCGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAACGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAATGTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTATGTAGATATACTAGAAGTTCTCCTCGACCGGTCGATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTT

yG_MATa :

AATTCATCTAGAGAAGAAAGCAAAGCCTTAATTCCAAGGAAAAAGAAGAAGTTGCAAAGAAATGTGGCATTACTCCACTTCAAGTAAGAGTTTGGGTATGTAATATGAGAATCAAACTTAAATATATCCTATACGTAGTATGGCGGAAAACATAAACAGAACTCTGTTTAACATTCTAGGTACTGAGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATCTAAAATTCCCGGGATCCGCTGTACGCGGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTGCGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATTTTAATAAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGCAGAAAAAGAAACAAGGAAGAAAGGGAACGAGAACAATGACGAGGAAACAAAAGATTAATAATTGCAGGTCTATTTATACTTGATAGCAAGACAGCAAACTTTTTTTTATTTCAAATTCAAGTAACTGGAAGGAAGGCCGTATACCGTTGCTCATTAGAGAGTAGTGTGCGTGAATGAAGGAAGGAAAAAGTTTCGTGTGCTTCGAGATACCCCTCATCAGCTCTGGAACAACGACATCTGTTGGTGCTGTCTTTGTCGTTAATTTTTTCCTTTAGTGTCTTCCATCATTTTTTTGTCATTGCGGATATGGTGAGACAACAACGGGGGAGAGAGAAAAGAAAAAAAAAGAAAAGAAGTTGTAAACCCACACCGGGTGTCATAATCAACCAATCGTAACTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAACAAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAGGGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCTTCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGTCCACTAGTCCCGGGAGCAAGATCAAGATGTTTTCACCGATCTTTCCGGTCTCTTTGGCCGGGGTTTACGGACGATGGCAGAAGACCAAAGCGCCAGTTCATTTGGCGAGCGTTGGTTGGTGGATCAAGCCCACGCGTAGGCAATCCTCGCAGATCTCGAACCATGTAATTTCCGAATACGGTAATTACACGCATCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCCTTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACACACAAACACAAATACACACACTAAAAAGCTT

yG_MATx1 :

AAGCTTGGATTCTCACAATCCTGTCGGTCACTTCTCGGCTGTTCGCGTATATTTTTTGTTGATACTTTTACCGGTATTTTGTCTGTAATTTATTCTCTATCACTGATAGGGACTTCTCTATCACTGATAGGGAACCCAGCCTGATTTATACTATTAGGGATCGCAGGAAGGCGGTGGGAAGTCCGGGAGTCGCTGAGGGGAAGTGTCAGTGGTTTTGGGTATAAATGGCTGGTTGTTCCCTATCAGTAATAGAGAATTCCCTATCAGTGATAGAGACTGCGGATTTAGAAACTACCTGATAAAAGTATCAACAAAAATTGCGCATGCCGGCCTGGATTTTGCGCAAATTTACCTTAACGTCCCACAATATGTTTACTTCGAAGCCTGCTTTCAAAATTAAGAACAAAGCATCCAAATCATACAGAAACACAGCGGTTTCAAAAAAGCTGAAAGAAAAACGTCTAGCTGAGCATGTGAGGCCAAGCTGCTTCAATATTATTCGACCACTCAAGAAAGATATCCAGATTCCTGTTCCTTCCTCTCGATTTTTAAATAAAATCCAAATTCACAGGATAGCGTCTGGAAGTCAAAATACTCAGTTTCGACAGTTCAATAAGACATCTATAAAATCTTCAAAGAAATATTTAAACTCATTTATGGCTTTTAGAGCATATTACTCACAGTTTGGCTCCGGTGTAAAACAAAATGTCTTGTCTTCTCTGCTCGCTGAAGAATGGCACGCGGACAAAATGCAGCACGGAATATGGGACTACTTCGCGCAACAGTATAATTTTATAAACCCTGGTTTTGGTTTTGTAGAGTGGTTGACGAATAATTATGCTGAAGTACGTGGTGACGGATATTGGGAAGATGTGTTTGTACATTTGGCCTTATAGAGTGTGGTCGTGGCGGAGGTTGTTTATCTTTCGAGTACTGAATGTTGTCAGTATAGCTATCCTATTTGAAACTCCCCATCGTCTTGCTGCAG

yG_MATx2 :

CTGCAGAGTAGTGTCTGAGGTACAAACATCTTAGTAGTGTCGAGAGGGTTGATTGTTTATGTATTTTTGCGAAATATATATATATATATTCTACACAGATATATACATATTTGTTTTTCGGGCTCATTCTTTCTTCTTTGCCAGAGGCTCACCGCTCAAGAGGTCCGCTAATTCTGGAGCGATTGTTATTGTTTTTTCTTTTCTTCTTCTATTCGAAACCCAGTTTTTGATTTGAATGCGAGATAAACTGGTATTCTTCATTAGATTCTCTAGGCCCTTGGTATCTAGATATGGGTTCTCGATGTTCTTTGCAAACCAACTTTCTAGTATTCGGACATTTTCTTTTGTAAACCGGTGTCCTCTGTAAGGTTTAGTACTTTTGTTTATCATATCTTGAGTTACCACATTAAATACCAACCCATCCGCCGATTTATTTTTCTGTGTAAGTTGATAATTACTTCTATCGTTTTCTATGCTGCGCATTTCTTTGAGTAATACAGTAATGGTAGTAGTGAGTTGAGATGTTGTTTGCAACAACTTCTTCTCCTCATCACTAATCTTACGGTTTTTGTTGGCCCTAGATAAGAATCCTAATATATCCCTTAATTCAACTTCTTCTTCTGTTGTTACACTCTCTGGTAACTTAGGTAAATTACAGCAAATAGAAAAGAGCTTTTTATTTATGTCTAGTATGCTGGATTTAAACTCATCTGTGATTTGTGGATTTAAAAGGTCTTTAATGGGTATTTTATTCATTTTTTCTTAGTGTGTGTATTTGTATTTGCGTGTCTATAGAAGTATAGTAATTTATGCTGCAAAGGTCCTAATGTATAAGGAAAAAAAATTTAGAGAAAAAAAGAAAAAAAGAGTTTTATATACATACAGAGCACATACATGCCATATAATCATGTATATACGCGCACATATATATATGCCTGTATGTGTCAGCACTAAATTTACCTGAACATACGCGCTATATATACGCGCCTCGCGTATATGCTCGAGGATTCCCTACGCGTGGGCTTTTTTTACTAACCAACGCGCGCGAAATACTAGT

MATα1 on pTet promoter :

GGGAGTCGCTGAGGGGAAGTGTCAGTGGTTTTGGGTATAAATGGCTGGTTGTTCCCTATCAGTAATAGAGAATTCCCTATCAGTGATAGAGACTGCGGATTTAGAAACTACCTGATAAAAGTATCAACAAAAATTGCGCATGCCGGCCTGGATTTTGCGCAAATTTACCTTAACGTCCCACAATATGTTTACTTCGAAGCCTGCTTTCAAAATTAAGAACAAAGCATCCAAATCATACAGAAACACAGCGGTTTCAAAAAAGCTGAAAGAAAAACGTCTAGCTGAGCATGTGAGGCCAAGCTGCTTCAATATTATTCGACCACTCAAGAAAGATATCCAGATTCCTGTTCCTTCCTCTCGATTTTTAAATAAAATCCAAATTCACAGGATAGCGTCTGGAAGTCAAAATACTCAGTTTCGACAGTTCAATAAGACATCTATAAAATCTTCAAAGAAATATTTAAACTCATTTATGGCTTTTAGAGCATATTACTCACAGTTTGGCTCCGGTGTAAAACAAAATGTCTTGTCTTCTCTGCTCGCTGAAGAATGGCACGCGGACAAAATGCAGCACGGAATATGGGACTACTTCGCGCAACAGTATAATTTTATAAACCCTGGTTTTGGTTTTGTAGAGTGGTTGACGAATAATTATGCTGAAGTACGTGGTGACGGATATTGGGAAGATGTGTTTGTACATTTGGCCTTATAG

α2 on CYC1 promoter :

ATTTCGCGCGCGTTGGTTAGTAAAAAAAGCCCACGCGTAGGGAATCCTCGAGCATATACGCGAGGCGCGTATATATAGCGCGTATGTTCAGGTAAATTTAGTGCTGACACATACAGGCATATATATATGTGCGCGTATATACATGATTATATGGCATGTATGTGCTCTGTATGTATATAAAACTCTTTTTTTCTTTTTTTCTCTAAATTTTTTTTCCTTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACACGCAAATACAAATACACACACTAAGAAAAAATGAATAAAATACCCATTAAAGACCTTTTAAATCCACAAATCACAGATGAGTTTAAATCCAGCATACTAGACATAAATAAAAAGCTCTTTTCTATTTGCTGTAATTTACCTAAGTTACCAGAGAGTGTAACAACAGAAGAAGAAGTTGAATTAAGGGATATATTAGGATTCTTATCTAGGGCCAACAAAAACCGTAAGATTAGTGATGAGGAGAAGAAGTTGTTGCAAACAACATCTCAACTCACTACTACCATTACTGTATTACTCAAAGAAATGCGCAGCATAGAAAACGATAGAAGTAATTATCAACTTACACAGAAAAATAAATCGGCGGATGGGTTGGTATTTAATGTGGTAACTCAAGATATGATAAACAAAAGTACTAAACCTTACAGAGGACACCGGTTTACAAAAGAAAATGTCCGAATACTAGAAAGTTGGTTTGCAAAGAACATCGAGAACCCATATCTAGATACCAAGGGCCTAGAGAATCTAATGAAGAATACCAGTTTATCTCGCATTCAAATCAAAAACTGGGTTTCGAATAGAAGAAGAAAAGAAAAAACAATAACAATCGCTCCAGAATTAGCGGACCTCTTGAGCGGTGAGCCTCTGGCAAAGAAGAAAGAATGA

STE12 on pTet promoter :

GGACTTCCCACCGCCTTCCTGCGATCCCTAATAGTATAAATCAGGCTGGGTTCCCTATCAGTGATAGAGAAGTCCCTATCAGTGATAGAGAATAAATTACAGACAAAATACCGGTAAAAGTATCAACAAAAAATATACGCGAACAGCCGAGAAGTGACCGACAGGATTGTGAGAATCCAAGCTTCCAAGGATGAAAGTCCAAATAACCAATAGTAGAACAGAGGAAATCTTAAAAGTTCAAGCTAATAATGAAAACGATGAAGTCAGTAAAGCTACTCCGGGCGAAGTTGAAGAATCGCTAAGGTTAATCGGCGATCTAAAATTCTTTTTAGCCACAGCGCCGGTAAATTGGCAAGAAAACCAAATTATAAGGCGATACTATCTGAATAGTGGACAAGGCTTTGTCTCTTGTGTATTTTGGAACAATCTATACTATATTACAGGTACTGATATTGTCAAATGTTGTCTTTACAGAATGCAAAAGTTTGGGAGAGAAGTAGTTCAAAAGAAAAAATTTGAAGAGGGTATTTTTTCAGATTTAAGAAATCTCAAATGTGGTATAGATGCAACTTTAGAACAACCAAAGTCCGAATTTTTGTCGTTTCTATTCAGAAATATGTGTCTGAAAACCCAAAAAAAGCAGAAAGTATTTTTTTGGTTCAGTGTAGCACATGATAAGTTGTTTGCGGATGCGTTGGAAAGAGATTTAAAAAGAGAAAGTTTGAATCAGCCTTCAACGACTAAGCCCGTTAATGAGCCCGCCTTATCTTTTTCATATGATTCCTCATCTGATAAGCCTCTCTACGATCAGTTACTTCAACATTTAGATTCTAGAAGACCATCTAGTACAACAAAATCAGATAATTCGCCTCCAAAATTAGAAAGCGAGAATTTTAAGGATAATGAGTTGGTAACAGTAACTAATCAGCCGCTTTTAGGCGTTGGCCTCATGGATGACGATGCGCCAGAATCCCCCTCTCAAATTAATGATTTTATTCCTCAGAAATTGATTATAGAACCCAATACTCTCGAATTGAATGGTCTCACAGAAGAAACGCCTCATGACTTACCCAAGAATACCGCTAAGGGCAGAGACGAAGAAGATTTTCCTCTCGACTATTTTCCTGTATCTGTTGAATACCCTACGGAGGAAAATGCGTTTGATCCGTTCCCTCCACAGGCTTTTACGCCAGCTGCCCCTTCCATGCCTATTTCCTATGATAACGTGAATGAAAGGGATTCTATGCCCGTTAATTCTCTTCTTAATAGATACCCCTATCAGTTATCAGTGGCACCCACTTTCCCAGTGCCACCATCATCATCGAGGCAACATTTTATGACAAATCGGGATTTTTATTCATCTAACAATAACAAGGAAAAATTGGTATCTCCTAGCGACCCTACCAGCTACATGAAGTATGACGAACCAGTTATGGATTTTGATGAATCTCGGCCAAATGAAAACTGTACAAATGCAAAATCTCACAACTCTGGCCAGCAAACTAAACAACACCAATTATATTCTAACAACTTCCAGCAATCTTACCCAAACGGAATGGTTCCAGGATACTACCCAAAAATGCCGTATAATCCCATGGGGGGGGATCCTCTACTCGATCAAGCCTTTTATGGCGCGGACGATTTTTTCTTTCCACCAGAAGGATGTGATAACAATATGCTGTATCCACAAACTGCAACTTCATGGAATGTTTTGCCCCCTCAAGCTATGCAACCAGCTCCAACCTATGTTGGGAGACCATACACACCGAATTATAGATCGACACCAGGTTCCGCGATGTTCCCATACATGCAAAGTTCAAATTCCATGCAGTGGAACACTGCTGTTTCACCTTATAGTTCGAGAGCACCATCTACAACTGCTAAAAACTATCCTCCTAGCACATTTTATTCTCAAAATATAAATCAATACCCACGGCGAAGAACTGTGGGAATGAAGTCTTCACAAGGAAATGTTCCAACAGGTAATAAACAATCTGTGGGCAAGTCTGCAAAAATTTCAAAGCCTCTACATATTAAGACAAGTGCTTATCAGAAGCAATACAAAATCAACTTGGAAACGAAAGCCAGGCCAAGTGCTGGTGACGAAGATTCTGCTCATCCTGATAAGAACAAAGAAATTTCGATGCCTACTCCGGATTCCAATACTTTGGTGGTCCAGTCAGAAGAAGGTGGAGCTCATTCACTTGAGGTAGATACCAATCGAAGGTCCGATAAAAACCTTCCAGATGCAACCTGATATAATATAATTTTTGAATTTATGATACAAGAATTAAAAATGCGGGCCAGAATTTAATATTAAACAATACTCAGAAGAAAACAACAAGGACAATCTGTTTTTATAAATAAAACAATCTTATACAAGACTAGAGCAAACAAAAAGCAAGAAAAAAAGGTAATAAATGTAACAAATCT

TetR on ADH1 promoter :

CAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTTATTAAAATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGCAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCGCGTACAGCGGATCCCGGGAATTTTAGATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTG

STE12 on a-specific CYC1 promoter :

CCCGGGAGCAAGATCAAGATGTTTTCACCGATCTTTCCGGTCTCTTTGGCCGGGGTTTACGGACGATGGCAGAAGACCAAAGCGCCAGTTCATTTGGCGAGCGTTGGTTGGTGGATCAAGCCCACGCGTAGGCAATCCTCGCAGATCTCGAACCATGTAATTTCCGAATACGGTAATTACACGCATCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCCTTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACACACAAACACAAATACACACACTAAAaagcttCCAAGGATGAAAGTCCAAATAACCAATAGTAGAACAGAGGAAATCTTAAAAGTTCAAGCTAATAATGAAAACGATGAAGTCAGTAAAGCTACTCCGGGCGAAGTTGAAGAATCGCTAAGGTTAATCGGCGATCTAAAATTCTTTTTAGCCACAGCGCCGGTAAATTGGCAAGAAAACCAAATTATAAGGCGATACTATCTGAATAGTGGACAAGGCTTTGTCTCTTGTGTATTTTGGAACAATCTATACTATATTACAGGTACTGATATTGTCAAATGTTGTCTTTACAGAATGCAAAAGTTTGGGAGAGAAGTAGTTCAAAAGAAAAAATTTGAAGAGGGTATTTTTTCAGATTTAAGAAATCTCAAATGTGGTATAGATGCAACTTTAGAACAACCAAAGTCCGAATTTTTGTCGTTTCTATTCAGAAATATGTGTCTGAAAACCCAAAAAAAGCAGAAAGTATTTTTTTGGTTCAGTGTAGCACATGATAAGTTGTTTGCGGATGCGTTGGAAAGAGATTTAAAAAGAGAAAGTTTGAATCAGCCTTCAACGACTAAGCCCGTTAATGAGCCCGCCTTATCTTTTTCATATGATTCCTCATCTGATAAGCCTCTCTACGATCAGTTACTTCAACATTTAGATTCTAGAAGACCATCTAGTACAACAAAATCAGATAATTCGCCTCCAAAATTAGAAAGCGAGAATTTTAAGGATAATGAGTTGGTAACAGTAACTAATCAGCCGCTTTTAGGCGTTGGCCTCATGGATGACGATGCGCCAGAATCCCCCTCTCAAATTAATGATTTTATTCCTCAGAAATTGATTATAGAACCCAATACTCTCGAATTGAATGGTCTCACAGAAGAAACGCCTCATGACTTACCCAAGAATACCGCTAAGGGCAGAGACGAAGAAGATTTTCCTCTCGACTATTTTCCTGTATCTGTTGAATACCCTACGGAGGAAAATGCGTTTGATCCGTTCCCTCCACAGGCTTTTACGCCAGCTGCCCCTTCCATGCCTATTTCCTATGATAACGTGAATGAAAGGGATTCTATGCCCGTTAATTCTCTTCTTAATAGATACCCCTATCAGTTATCAGTGGCACCCACTTTCCCAGTGCCACCATCATCATCGAGGCAACATTTTATGACAAATCGGGATTTTTATTCATCTAACAATAACAAGGAAAAATTGGTATCTCCTAGCGACCCTACCAGCTACATGAAGTATGACGAACCAGTTATGGATTTTGATGAATCTCGGCCAAATGAAAACTGTACAAATGCAAAATCTCACAACTCTGGCCAGCAAACTAAACAACACCAATTATATTCTAACAACTTCCAGCAATCTTACCCAAACGGAATGGTTCCAGGATACTACCCAAAAATGCCGTATAATCCCATGGGGGGGGATCCTCTACTCGATCAAGCCTTTTATGGCGCGGACGATTTTTTCTTTCCACCAGAAGGATGTGATAACAATATGCTGTATCCACAAACTGCAACTTCATGGAATGTTTTGCCCCCTCAAGCTATGCAACCAGCTCCAACCTATGTTGGGAGACCATACACACCGAATTATAGATCGACACCAGGTTCCGCGATGTTCCCATACATGCAAAGTTCAAATTCCATGCAGTGGAACACTGCTGTTTCACCTTATAGTTCGAGAGCACCATCTACAACTGCTAAAAACTATCCTCCTAGCACATTTTATTCTCAAAATATAAATCAATACCCACGGCGAAGAACTGTGGGAATGAAGTCTTCACAAGGAAATGTTCCAACAGGTAATAAACAATCTGTGGGCAAGTCTGCAAAAATTTCAAAGCCTCTACATATTAAGACAAGTGCTTATCAGAAGCAATACAAAATCAACTTGGAAACGAAAGCCAGGCCAAGTGCTGGTGACGAAGATTCTGCTCATCCTGATAAGAACAAAGAAATTTCGATGCCTACTCCGGATTCCAATACTTTGGTGGTCCAGTCAGAAGAAGGTGGAGCTCATTCACTTGAGGTAGATACCAATCGAAGGTCCGATAAAAACCTTCCAGATGCAACCTGATATAATATAATTTTTGAATTTATGATACAAGAATTAAAAATGCGGGCCAGAATTTAATATTAAACAATACTCAGAAGAAAACAACAAGGACAATCTGTTTTTATAAATAAAACAATCTTATACAAGACTAGAGCAAACAAAAAGCAAGAAAAAAAGGTAATAAATGTAACAAATCT

Parts of a1 ORF :

GAAGAAAGCAAAGCCTTAATTCCAAGGAAAAAGAAGAAGTTGCAAAGAAATGTGGCATTACTCCACTTCAAGTAAGAGTTTGGGTATGTAATATGAGAATCAAACTTAAATATATCCTATACGTAGTATGGCGGAAAACATAAACAGAACTCTGTTTAACATTCTAGGTACTGAG

Appendix

Personnel

Hynek Kasl - Responsible person

Anna Sosnová - Experimental assistance

Václav Pelíšek - Experimental assistance

Tereza Puchrová - Scientific advisor

Useful Links

Protocols page:

Protocols

Old Links

See following pages for detailed information about constructing the synthetic haploids:

• MATα
• MATa

References

1. ↑ Zhong, H., McCord, R., & Vershon, a K. (1999). Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome. Genome Research, 9(11), 1040–1047.
2. ↑ Curran, K. A., Crook, N. C., Karim, A. S., Gupta, A., Wagman, A. M., & Alper, H. S. (2014). Design of synthetic yeast promoters via tuning of nucleosome architecture. Nat Commun, 5. Retrieved from http://dx.doi.org/10.1038/ncomms5002
3. ↑ Ellis, T., Wang, X., & Collins, J. J. (2009). Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nature Biotechnology, 27(5), 465–471. http://doi.org/10.1038/nbt.1536
4. ↑ Pi H, Chien CT, Fields S. Transcriptional activation upon pheromone stimulation mediated by a small domain of Saccharomyces cerevisiae Ste12p. Molecular and Cellular Biology. 1997;17(11):6410-6418.