G-DIG/PRO-V plasmid construction

G-DIG/PRO-V fusion constructs were prepared using the Gibson method (Gibson et al., 2009). Constructs were cloned into the plasmid p416CYC (p16C). Gal4 (residues 1–93, UniProt ID P04386), DIG10.3 (Tinberg et al., 2013), and VP16 (residues 363-490, UniProt ID P06492) PCR products for were amplified from their respective templates using Phusion high-fidelity polymerase (NEB, Waltham, MA) and standard PCR conditions (98°C 10 s, 60°C 20 s, 72°C 30 s; 30 cycles). The 8-residue linker sequence GGSGGSGG was used between Gal4 and DIG10.3. PCR primers were purchased from Integrated DNA technologies and contained 24–30 5’ bases of homology to either neighboring fragments or plasmid. Clones containing an N-terminal degron were similarly cloned fusing residues 1–67 of Matα2 (UniProt ID P0CY08) to the 5’- end of G-DIG-V. Plasmids were transformed into yeast using the Gietz method (Gietz and Schiestl, 2007), with transformants being plated on synthetic complete media lacking uracil (SD -ura).