Transformations were performed with a standard lithium acetate method. To construct Z3EV and Z4EV strains, the Gal4dbd of GEV was deleted by PCR-mediated disruption with URA3. Zif268 and Z4 DNA-binding domains with homology to the ER and ACT1 promoter were then transformed into cells and selected via 5-Fluoroorotic Acid (5-FOA) counter selection of URA3. Single colonies were isolated and sequenced to verify the presence of the proper DNA-binding domain (Supplementary Figure S1A).
The reporter plasmid was created from the base plasmid pRS416 (gift from Megan McClean), a CEN plasmid containing the URA3 selectable marker. The GAL1 promoter region was amplified from genomic DNA. Overlap-extension PCR was used to add the restriction enzyme sites for XbaI and NotI, respectively, on either side of the region of the three continuous Gal4p-binding sites 5′-CGG-N11-CCG-3′ (22 ). This promoter fragment was then cloned into pRS416 in front of green fluorescent protein (GFP) using the restriction enzymes NheI and XmaI. Before zinc-finger-binding sites were added the XbaI site in GFP was re-coded using a silent mutation to remove this restriction site. Finally, the three canonical Gal4p-binding sites were removed via digestion with XbaI and NotI and triplets of dimeric Z3EV and Z4EV-binding sites were cloned into respective plasmids (Supplementary Data and Supplementary Figure S1B).
To construct the inducible GCN4 allele, KanMX-Z4EVpr was amplified from pMN10 with the primers 5′-caatttgtctgctcaagaaaataaattaaatacaaataaaCGCACTTAACTTCGCATCTG-3′ and 5′-tggatttaaagcaaataaacttggctgatattcggacatTATAGTTTTTTCTCCTTGACG-3′ and transformed into Z4EV-containing parent strain. The uppercase portions of the sequences share homology with the KanMX-Z4EVpr cassette on the plasmid pMN10.