Mutants of the potential transcription factor binding sites were generated by overlap extension PCR (Sambrook et al., 1989). Oligonucleotides in which the potential transcription factor binding sites were mutated (Table 1b) were synthesized by Integrated DNA Technologies (Coralville, IA). In the first step of the PCR, two fragments were generated such that they overlap at the mutated sequence. Products were gel extracted and 75 ng of each product was used as template in the extension step. Products with overlapping ends were mixed with R-Taq polymerase and ten PCR cycles were run without primers. HE3629 and HE3636 were then added and PCR was continued for another 25 cycles to amplify the full-length fragment. Products of the extension step were column purified and cloned into BamHI and SalI sites in the pXP2 vector, upstream of the ADH4 promoter.
