The TET-OP2 promoter was inserted upstream of the endogenous FOB1 locus by one-step integration of a PCR fragment generated using primers FOB1tetF and FOB1tetR with pKAN-TETO2 as a template. The TETR'-SSN6 cassette was inserted by one-step integration into the met15Δ0 locus by digesting plasmid pLMI-tetR'S with PacI. The LEU2 marker was subsequently exchanged for ADE2 or MET15 using the primers LEU2swapF and LEU2swapR with the appropriate plasmid templates (pRS402 and pRS401, respectively [63]). The VP16 activation domain from the tTA activator was replaced with activation domain A of GCN4 by generating a GCN4A PCR product using GCN4salAF and GCN4ascAR primers with plasmid pGCN4 (a gift from S. Hahn) as a template. The PCR product was digested with SalI and AscI and subcloned into pUI-tTA-ADH1term-URA3 digested with the same enzymes to create pUI-tTA-GCN4A-ADH1term-URA3. The tTA-GCN4A-ADH1term-URA3 cassette was integrated into a neutral site on Chromosome I (coordinates 17030-17205) by one-step integration of a PCR product using the primers URA3-tTA-intCHRIF and URA3-tTA-intCHRIR. Subsequent strain construction to generate haploid and diploid MEP strains with the appropriate genotypes was performed by standard methods.
