Team:Czech Republic/Project/Synthetic haploids
Synthetic Haploids
Contents
Abstract
To use the mating pheromones as signals for multicellular communication, synthetic haploids of both mating types that preserve the ability to process an extracellular signal via pheromone response pathway even after mating - in a diploid state are needed. However, all components of the pathway are switched-off naturally in diploid cells, therefore synthetic haploid strains that mate into a diploid with a functional pheromone pathway were designed. As a result, the IODs can use the robust natural signaling pathway for signal transduction.
Key Achievements
- Constructed a set of reporter promoters for yeast cells
- Characterized reporter promoters in all mating types
- Designed and materialized synthetic MATa and MATx strains
- Built a synthetic diploid strain with a functional yeast pheromone pathway
- Demonstrated the correct functionality of yeast pheromone pathway in synthetic diploids
Yeast pheromone pathway
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
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.
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 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).
- 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).
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].
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.
- 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).
- 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.
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:
| Promoter | Template | Primer | Primer sequence |
|---|---|---|---|
| pADH1 | Yeast genome | pAHD1-F | ACATCACTCGAGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCC |
| pADH1-R | CTGAGTAAGCTTAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGC | ||
| asCYC1 | yG_MATa_2 | asCYC1-F | TAGCACCTCGAGCCCGGGAGCAAGATCAAGAT |
| asCYC1-R | AGACATAAGCTTTATTAATTTAGTGTGTGTATTTGTGTTTGTGT | ||
| pFUS1 | Yeast genome | pFUS1-F | TAGGGCCTCGAGTAATAATCAGAACTCCAACAATAGTCAACA |
| pFUS1-R | AGACATAAGCTTTTTGATTTTCAGAAACTTGATGGCT | ||
| pSTE2 | Yeast genome | pSTE2-F | TAGGGCCTCGAGATCCAATATCACCTGACCTTCATC |
| pSTE2-R | AAGCTTGAATTCTTTTGATTCTTGGATATGGTTCTTAACGGT | ||
| pSTE5 | Yeast genome | pSTE5-F | TAGGGCCTCGAGTCAAAGCAGTTTGTGCGATTTG |
| pSTE5-R | AAGCTTGAATTCTTAAAAGTTGTTTCCGCTGTATCC | ||
| pTv3 | yG_pTv3pTv3 | pTv3-F | TAGGGCCTCGAGGGACTTCCCACCGCCTTC |
| pTv3-R | AGACATAAGCTTGGATTCTCACAATCCTGTCGG |
All promoters were amplified in a single PCR run, with the following conditions:
| Property | Value |
|---|---|
| Polymerase | Q5 |
| Extension Time | 90s |
| Annealing temperature | 58°C |
| Number of cycles | 35 |
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
| Promoter | Enzyme 1 | Enzyme 2 |
|---|---|---|
| pADH1 | XhoI | HindIII |
| asCYC1 | XhoI | HindIII |
| pFUS1 | XhoI | HindIII |
| pSTE2 | XhoI | EcorI |
| pSTE5 | XhoI | EcorI |
| pTv3 | XhoI | HindIII |
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
| Part | 5' Enzyme | 3' Enzyme |
|---|---|---|
| yG_MATa | EcorI | HindIII |
| yG_MATx_1 | HindIII | PstI |
| yG_MATx_2 | PstI | SpeI |
| pRSII405(MATa) | EcorI | HindIII |
| pRSII405(MATx) | SpeI | HindIII |
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
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
In the end, we had two versions of synthetic haploids that differ only in the integration plasmid's marker - URA3 (pRSII406) or LEU2 (pRS405). We integrated the constructs into the genomes of MATa and MATx strain, 6193 and 6194, respectively. We mated the synthetic strains according to the schematic, positive and negative controls are included.
The plate looks as expected, both positive controls (mated wil-type MATa and MATx strains) grew (the left bottom only a little bit). All the negative controls (empty crosses) did not grow at all, and the synthetic strains mate (both the URA3 and LEU2 versions).
Reporter plasmids' characterisation
Materials and methods
Used strains
- Ecoli strain E5alpha
- Yeast strain 7283 MATx ura3
- Yeast strain 7284 MATa ura3
- Yeast strain 10150 MATa/x ura3/ura3
- Yeast strain 6193 MATa ura3 leu2 his3
- Yeast strain 6194 MATx ura3 leu2 trp1
Used material
- LB-M agar plates with chloramphenicol
- LB-M agar plates with ampicillin
- 1.5 ml eppendorf tubes
- 0.5 ml PCR tubes
- 50 ml centrifuge conical base and rim tubes
- NucleoSpin Plasmid DNA, RNA, and protein purification Kit
- NucleoSpin Gel and PCR Clean-up Kit
- LB-M medium with chloramphenicol
- NaOH agarose gel and buffer
- Sphero Rainbow Calibration Particles, 8 Peaks, 3.0-3.4
Used methods
- Transformation
- Miniprep
- Restriction digest
- Ligation
- NucleoSpin Gel Clean-up
- NucleoSpin Plasmid DNA purification
- Flow cytometry
All used protocols can be found here:
Protocols
Used software
- CFlow Plus
- Microsoft Excel
- 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:
Old Links
See following pages for detailed information about constructing the synthetic haploids:
References
- ↑ 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.
- ↑ 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
- ↑ 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
- ↑ 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.