MATα integration plasmid

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

'Wild-type MATα' 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.

Wild-type MATα locus

Integrated parts

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 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. Promoter pTet has sequence taken from [http://www.nature.com/nbt/journal/v27/n5/abs/nbt.1536.html here] (the T16 version). This promoter is repressed by TetR (tetracycline) and is active in absence of TetR (see this wikipeadia 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. Promoter CYC1 has sequence taken from [http://www.nature.com/ncomms/2014/140527/ncomms5002/full/ncomms5002.html this page] (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 ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Therefore, no synthetic terminator was included. Promoter pTet has sequence taken from [http://www.nature.com/nbt/journal/v27/n5/abs/nbt.1536.html here] (the T15 version).