MATa integration plasmid

MATα 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

• TetR on ADH1 promoter

Tetracycline repressor ORF sequence was taken from Addgene databese, the ADH1 promoter sequence was taken from same page. CYC1 terminator is included after TetR, it’s sequence was also taken from this page. Show sequence

• STE12 on a-specific CYC1

ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Sequence of a-specific CYC1 promoter was obtained from Prof. Vershon, who presented series of a-specific promoters^{[Vershon1999]} ihttp://www.ncbi.nlm.nih.gov/pubmed/10568744. It is a CYC1 promoter with inserted α2-Mcm1 binding sequence from AGA2 gene (α2-Mcm1 complex represses a-sg). Show sequence

• Parts of a1 ORF

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

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

References

1. ↑ Hualin Zhong, Ron McCord, and Andrew K. Vershon. Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome. doi: 10.1101/gr.9.11.1040 Genome Res. 1999. 9: 1040-1047 Cold Spring Harbor Laboratory Press