Table 7 Oligonucleotide primers used in this study
Name | Sequence (5′ → 3′) |
|---|---|
Primers for CRISPR-Cas plasmid assembly | |
 Plasmid backbone amplification | GATCATTTATCTTTCACTGCGGAGAAG |
 ARO10 gRNA CRISPR KO sequence | TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCATTTACAAGTATTCTAAACCGTTTTAGAGCTAGAAATAGCAAGTTA AAATAAGGCTAGTCCGTTATCAAC |
 ARO10 repair fragment upper | ACAAGTTGACGCGACTTCTGTAAAGTTTATTTACAAGATAACAAAGAAACTCCCTTAAGCAAACTTGTGGGCGCAATTATAAAACACTGCTACCAA TTGTTCGTTTTCTGTTCATTAACA |
 ARO10 repair fragment lower | TGTTAATGAACAGAAAACGAACAATTGGTAGCAGTGTTTTATAATTGCGCCCACAAGTTTGCTTAAGGGAGTTTCTTTGTTATCTTGTAAATAAACT TTACAGAAGTCGCGTCAACTTGT |
Primers for verification of knockouts | |
 PDC1 upstream fwd | AGCTGTCCTCGTTGAACATAG |
 PDC1 downstream rev | TTGCGTGAGGTTATGAGTAG |
 PDC5 upstream fwd | CAGAACCACCTACACTACC |
 PDC5 downstream rev | CTGGGTTCTTAGCATCCTTG |
 PDC6 upstream fwd | AACTCCCGCAAACAAAGGTG |
 PDC6 downstream rev | CAACACCTGCGAGATACCGTAG |
 Aro10 upstream Fwd | TGCTTGTACACCTCATGTAG |
 Aro10 downstream Rev | GCAGACATTTAGCAGATGTAG |
Primers for verification of plasmid assembly and transformation | |
 GPD1 promoter Fwd | GGGATGTGCTGCAAGGCGATTAAGTTGG |
 CYC1 terminator Rev | GGCAGTGAGCGCAACGCAATTAATGTGAG |
Primers for verification of genome integrations | |
 MTH1ΔT conformation fwd | CACCATGTTTGTTTCACCACCACCAGCAACTTCG |
 MTH1ΔT conformation rev | TCAGGATACTGAATCCGGCTGCCAATCCA |
 PDC1-AmdS at ADE2 conformation fwd | ATGTTATGCGCCTGCTAGAG |
 PDC1-AmdS at ADE2 conformation rev | ACATTCCGCCATACTGGAGG |
 Cas9-tag-natNT2 at PDC6 conformation fwd | AACTCCCGCAAACAAAGGTG |
 Cas9-tag-natNT2 at PDC6 conformation rev | CAACACCTGCGAGATACCGTAG |
Primers for plasmid construction | |
 KdcA fwd GPDP homology | CTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAGAACTTAGTTTCGACGGATATGGATACAGTAGGAGATTACCTGTTAGACCG |
 KdcA rev CYC1T homology | CGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGACTATTTATTTTGCTCAGCAAATAATTTACCC |
 KivD fwd GPDP homology | CTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAGAACTTAGTTTCGACGGATATGTACACTGTTGGTGACTAC |
 KivD rev CYC1T homology | CGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATTAAGACTTGTTTTGTTCAGCGAACAACTTACCC |
Primers for cassette construction | |
 MTH1ΔT fwd | CACCATGTTTGTTTCACCACCACCAGCAACTTCG |
 MTH1ΔT rev | TCAGGATACTGAATCCGGCTGCCAATCCA |
 PDC1-ADE2 homology fwd | TTGCCCCAAGGCCTCACAACTCTGGACATTATACCATTGATGCTTGCGTCACTTCTCAATTTGAAGCTCATTTGAGATCAATATTGGATTTGCCAA TGCCTGCGACTGGGTGAGCATATG |
 PDC1-TEF2T homology rev | CAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGATCGGTTTTTTTTTTGGAAGACATCTTTTCC |
 AmdS cassette fwd | CAGTATAGCGACCAGCATTC |
 AmdS-ADE2 homology rev | GGCATTGGCAAATCCAATATTGATCTCAAATGAGCTTCAAATTGAGAAGTGACGCAAGCATCAATGGTATAATGTCCAGAGTTGTGAGGCCTTG GGGCAAGACATGGAGGCCCAGAATAC |
 PDC1-AmdS-ADE2 homology fwd | TCTAAGTACATCCTACTATAACAATC |
 PDC1-AmdS-ADE2 homology rev | CATTTGATGTAATCATAACAAAGCC |
 Cas9-PDC6 homology fwd | GTGCCTATTGATGATCTGGCGGAATGTCTGCCGTGCCATAGCCATGCCTTCACATATAGTCCGCAAATTAAAGCCTTCGAG |
 Cas9-tag homology rev | GCAGTCCTCTTTTATATACAGTATAAATAAAAAACCAGTAATATAGCAAAAACATATTGCCAGGGAACAAAAGCTGGAGCTCATAG |
 natNT2-tag homology fwd | GTGCCTATTGATGATCTGGCGGAATGTCTGCCGTGCCATAGCCATGCCTTCACATATAGTCCGCAAATTAAAGCCTTCGAG |
 natNT2-PDC6 homology rev | CAAACTGTGTAAATTTATTTATTTGCAACAATAATTCGTTTTTGAGTACACTACTAATGGCATAGGCCACTAGTGGATCTG |