Methods S1. Sequences relevant to the constructed strains, Related to Figures 1-6. A. Promoter Sequences Gal4 binding sites are highlighted in the color referenced in Figure 1A when possible. Site 1: red, Site 2: grey, Site 3: green, Site 4: blue. Note that the second binding site has been changed from orange in Figure 1A to grey here. Also note that these positions are reversed for the GAL10 promoter. Site 1: blue, Site 2: green, Site 3: grey, Site 4: red. Base Pairs which differ from the wild type are written in capital letters. Base pairs which have been removed are highlighted in black with white text. Parentheses indicate where the sequence can be located. GE1: Wild Type GAL1 promoter sequence (GenBank ID code: MH888036) GE105: Wild Type GAL10 promoter sequence (GenBank ID code: MH888037) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE6: CRISPR cut site replacing the endogenous GAL1/GAL10 promoter (GenBank ID code: MH888038) cagccaaacgaccaggtgcgcttccgggga
GE117: Base pairs 4-8 5 of 1 st Gal4 binding site recoded (GenBank ID code: MH818048) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatagctaggtacggattagaagccgcc GE118: Base pairs 7-15 5 of 1 st Gal4 binding site recoded (GenBank ID code: MH818049) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattaagcgttcgaagtacggattagaagccgcc GE123: Endogenous GAL1/GAL10 promoter edited to remove GAL10 transcription due to loss of the 4 th Gal4 binding site (GenBank ID code: MH818050) atgtgccgatgcacacgctctggtgcaatacaaagattctacaatactagcttttatggttatgaagaggaaaaatt
GE124: Endogenous GAL1/GAL10 promoter edited to remove GAL1 transcription due to loss of 5 regulatory regions (GenBank ID code: MH818051) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctataagtagattcggattagaagccgcc GE135: Base pairs 7-15 5 of 1 st Gal4 binding site recoded (GenBank ID code: MH818052) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcgaa CGCTTAatgagcagttaagcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctcttt GE140: TAG within 1 st Gal4 binding site changed to CCA (GenBank ID code: MH818053) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtacggatccaaagccgcc
GE149: ATT within 1 st Gal4 binding site changed to GCG (GenBank ID code: MH818054) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtacgggcgagaagccgcc GE151: AAAG 6 base pairs 3 of 4 th Gal4 binding site changed to CGGC (GenBank ID code: MH818055) atgtgcctcgcgccgcactgctccgaacaatcggcattctacaatactagcttttatggttatgaagaggaaaaatt GE153: TCTCCT within 3 rd Gal4 binding site changed to AAACCG (GenBank ID code: MH818056) gagcgggcgacagccctccgacggaagacaaaccgccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag
GE155: 11bp deleted between 3 rd and 4 th Gal4 binding sites (GenBank ID code: MH818057) GE156: 21bp deleted between 3 rd and 4 th Gal4 binding sites (GenBank ID code: MH818058) GE163: AT 3 of the 4 th Gal4 binding site changed to GG (GenBank ID code: MH818059) atgtgcctcgcgccgcactgctccgaacaataaagggtctacaatactagcttttatggttatgaagaggaaaaatt
GE164: CGG of the 1 st Gal4 binding site changed to TTT (GenBank ID code: MH818060) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtatttattagaagccgcc GE165: CCG of the 1 st Gal4 binding site changed to TTT (GenBank ID code: MH818061) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtacggattagaagccgtt Tagcgggcgacagccctccgacggaagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag GE166: AGCA 3 of the 4 th Gal4 binding site changed to TTTT (GenBank ID code: MH818062) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat atttttatgagcagttaagcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctcttt
GE168: CTTT 5 of the 1 st Gal4 binding site changed to GCCG (GenBank ID code: MH818063) agctagtattgtagaatgccgattgttcggagcagtgcggcgcgaggcacatctgcgtttcaggaacgcgaccggtg aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE170: AGGAGA within 2 nd Gal4 binding site changed to CGGTTT (GenBank ID code: MH818064) aagacgaggacgcacggcggtttgtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE182: CGC within 4 th Gal4 binding site changed to TTT (GenBank ID code: MH818065) atgtgccttttgccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaatt
GE183: CCG within 4 th Gal4 binding site changed to TTT (GenBank ID code: MH818066) atgtgcctcgcgccgcactgcttttaacaataaagattctacaatactagcttttatggttatgaagaggaaaaatt GE184: Interior of 2 nd Gal4 binding site recoded (GenBank ID code: MH818067) gagcggccttaaaacctccgacggaagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag GE185: AA within the 3 rd Gal4 binding site changed to CG (GenBank ID code: MH818068) gagcgggcgacagccctccgacggcggactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag
GE186: GAC within the 3 rd Gal4 binding site changed to TTT GenBank ID code: (MH818069) gagcgggcgacagccctccgacggaattttctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag GE187: TCT within the 3 rd Gal4 binding site changed to AAG (GenBank ID code: MH818070) gagcgggcgacagccctccgacggaagacaagcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag GE188: CCT within the 3 rd Gal4 binding site changed to AAA (GenBank ID code: MH818071) gagcgggcgacagccctccgacggaagactctaaaccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag
GE190: CTA within 4 th Gal4 binding site changed to TGG (GenBank ID code: MH818072) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggctttggatccgtacttcaat GE192: TA 5 of the 1 st Gal4 binding site changed to CC (GenBank ID code: MH818073) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaac Ctagcaatgagcagttaagcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctcttt GE193: CCG of the 4 th Gal4 binding site changed to AAA (GenBank ID code: MH818074) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaataaatacttcaat
GE194: CGG of the 4 th Gal4 binding site changed to AAA (GenBank ID code: MH818075) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctaaacggcttctaatccgtacttcaat GE195: TA 3 of the 4 th Gal4 binding site changed to GG (GenBank ID code: MH818076) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GGagcaatgagcagttaagcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctcttt GE196: CGG within 1 st Gal4 binding site changed to AAA (GenBank ID code: MH818077) agctagtattgtagaatctttattgttaaaagcagtgcggcgcgaggcacatctgcgtttcaggaacgcgaccggtg aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat
GE197: Interior of 3 rd Gal4 binding site recoded (GenBank ID code: MH818078) aagacgaggacgcacggaggagagtcttccgtcggaggttttaaggccgctcggcggcttctaatccgtacttcaat GE200: TT within the 2 nd Gal4 binding site changed to GC (GenBank ID code: MH818079) aagacgaggacgcacggaggagagtcgcccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE201: GTC within the 2 nd Gal4 binding site changed to AAA (GenBank ID code: MH818080) aagacgaggacgcacggaggagaaaattccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat
GE202: AGA within the 2 nd Gal4 binding site changed to CTT (GenBank ID code: MH818081) aagacgaggacgcacggaggcttgtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE203: AGG within the 2 nd Gal4 binding site changed to TTT (GenBank ID code: MH818082) aagacgaggacgcacggtttagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE204: GCG within 1 st Gal4 binding site changed to AAA (GenBank ID code: MH818083) agctagtattgtagaatctttattgttcggagcagtgcggcaaaaggcacatctgcgtttcaggaacgcgaccggtg aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat
GE206: 11bp recoded between 3 rd and 4 th Gal4 binding sites (GenBank ID code: MH818084) gagcgggcgacagccctccgacggaagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcagtcccatact GE207: 7 th bp within the 3 rd Gal4 binding site changed from A to C (GenBank ID code: MH818085) gagcgggcgacagccctccgacggaagcctctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag GE208: 8 th bp within the 3 rd Gal4 binding site changed from C to A (GenBank ID code: MH818086) gagcgggcgacagccctccgacggaagaatctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag
GE209: 9 th bp within the 3 rd Gal4 binding site changed from T to G (GenBank ID code: MH818087) gagcgggcgacagccctccgacggaagacgctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag GE210: GTA immediately 5 of 1 st Gal4 binding site changed to TGC (GenBank ID code: MH818088) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaatgccggattagaagccgcc GE215: 11 th bp within 2 rd Gal4 binding site changed from T to G (GenBank ID code: MH818089) aagacgaggacgcacggaggagaggcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat
GE216: 10 th bp within 2 nd Gal4 binding site changed from G to T (GenBank ID code: MH818090) aagacgaggacgcacggaggagattcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE217: 9 th bp within 2 nd Gal4 binding site changed from A to C (GenBank ID code: MH818091) aagacgaggacgcacggaggagcgtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaat GE218: TAC immediately downstream of the 4 th Gal4 binding site changed to GCA (GenBank ID code: MH818092) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccggcattcaat
GE229: CRISPR cut site and YFP replacing the endogenous GAL1 gene and promoter (Cut site not highlighted, YFP in yellow) (GenBank ID code: MH888039) GGGTCCCGGGCAAAGGGCGCGTGATCGCGGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGT TGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAAC TTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGT CTAAAATGCTTTGCTAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGT ACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCC TTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC TATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAA CATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTAC CAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTT GAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAA GE230: Wild type GAL1 promoter in the ho locus with native GAL promoter knocked out (GenBank ID code: MH818093) ho: GE231: Edited GAL1 promoter with GAC within the 3 rd Gal4 binding site changed to TTT in the ho locus with native GAL promoter knocked out (GenBank ID code: MH818094) ho: gagcgggcgacagccctccgacggaattttctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcag
GE234: Wild type GAL1 promoter in native GAL locus driving YFP (GenBank ID code: MH818095) GE235: Edited GAL1 promoter with GTA immediately 5 of 1 st Gal4 binding site changed to TGC in native GAL locus driving YFP (GenBank ID code: MH818096) aaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaatgccggattagaagccgcc GE236: Edited GAL10 promoter with TAC immediately downstream of the 4 th Gal4 binding site changed to GCA in native GAL locus driving YFP (GenBank ID code: MH818097) aagacgaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccggcattcaat
GE241: All three RSC binding sites recoded to AAAA (GenBank ID code: MH818098) gagcgggcgacagccctccgacggaagactctcctccgtgcgtcctcgtcttcaccggtaaaattcctgaaaaaaag atgtgcctaaaaccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaatt B. grna cassette sequence (GenBank ID code: KT876200) tctttgaaaagataatgtatgattatgctttcactcatatttatacagaaacttgatgttttctttcgagtatatac aaggtgattacatgtacgtttgaagtacaactctagattttgtagtgccctcttgggctagcggtaaaggtgcgcat tttttcacaccctacaatgttctgttcaaaagattttggtcaaacgctgtagaagtgaaagttggtgcgcatgtttc ggcgttcgaaacttctccgcagtgaaagataaatgatcnnnnnnnnnnnnnnnnnnnngttttagagctagaaatag caagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttttttgttttttatg tct