TABLE 4.

Oligonucleotide primers used in this study

Primer ID	Sequence	Product(s)a
6005	GATCATTTATCTTTCACTGCGGAGAAG	gRNA pROS plasmid backbone amplification
6006	GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC	gRNA pROS plasmid backbone amplification
14229	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCAGTAGAATTTCACCTAGACGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for SynPAM gRNA plasmid
13686	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCCTGCGGTGATAGAACCCTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for ABZ1 gRNA plasmid
14988	CTTTTACACGATGACCTTTCGAGATTTCACAAGGGGGATAAAGGAAGTAGAATTTCACCTAGACGTGGATATTTGTATATTATTAGATATGTATGCAAACATTTTCTTTAGAA	ABZ1 KO repair oligonucleotide
14989	TTCTAAAGAAAATGTTTGCATACATATCTAATAATATACAAATATCCACGTCTAGGTGAAATTCTACTTCCTTTATCCCCCTTGTGAAATCTCGAAAGGTCATCGTGTAAAAG	ABZ1 KO repair oligonucleotide
13693	AAACCGCGAATATATAAAAACAAGC	ABZ1 mutant allele amplification
13694	GGCACAAAACGTCATTTTCC	ABZ1 mutant allele amplification
15075	TAATCACTCGGCAATGTGGAATTGTTACCGTGATAGCCTTCATGCAGTAGAATTTCACCTAGACGTGGATCTTATACCAATTTTATGCAGGATGCTGAGTGCTATTTGTTAGC	ARO7 KO repair oligonucleotide
15076	GCTAACAAATAGCACTCAGCATCCTGCATAAAATTGGTATAAGATCCACGTCTAGGTGAAATTCTACTGCATGAAGGCTATCACGGTAACAATTCCACATTGCCGAGTGATTA	ARO7 KO repair oligonucleotide
12052	CAGGAGTCTCTGAGCAAGGC	ARO7 mutant allele amplification
12053	ACCATGCTAAGAGCTGCTCC	ARO7 mutant allele amplification
15037	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCAGCATCAGAAGTAATAACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for BAS1 gRNA plasmid
15584	AAACTTTTGTTGTAGCGTTTTTGCTTTTTTTTTTTTATCGCAGAATACATTTTATCGAGATAGGTCTAGAGATCTGTTTAGCTTGC	Repair fragment with hphNT1 for BAS1 KO
15585	ATTACAAAACTAATATGTTAAACAATTGAAAGATTTGTGTTTTTTTTCGGCCTTGCCTTCAGCTCCAGCTTTTGTTCCC	Repair fragment with hphNT1 for BAS1 KO
13687	CCTTTGACGATGTGCAACGG	Amplification BAS1 mutant allele
13688	AACGCCCTTTGTGTTTGTGG	Amplification BAS1 mutant allele
13520	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCTCTTGCTGGACGTATAATGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for CNB1 gRNA plasmid
13612	ACTCAATGGTGATCAGAATCCATAGAAGCATTTTTATTTCTTAAAAGTAGAATTTCACCTAGACGTGGGACTAGGGGACACTTCATTCATTTATGGTATGCCAATATTTTTAA	CNB1 KO repair oligonucleotide
13613	TTAAAAATATTGGCATACCATAAATGAATGAAGTGTCCCCTAGTCCCACGTCTAGGTGAAATTCTACTTTTAAGAAATAAAAATGCTTCTATGGATTCTGATCACCATTGAGT	CNB1 KO repair oligonucleotide
13523	GCATCAGCACTGCAGAATCG	CNB1 mutant allele amplification
13524	GATCCCCCTTTGTGCATTGC	CNB1 mutant allele amplification
13521	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCCATAAAAAGAGAGACCACTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for PMR1 gRNA plasmid
13541	CCAGCACAGACGTAAGCTTAAGTGTAAGTAAAAGATAAGATAATTAGTAGAATTTCACCTAGACGTGGTATGTCACATTTTGTGCTTTTATCGTTTTTCCTTCCTTCCCTTTA	PMR1 KO repair oligonucleotide
13542	TAAAGGGAAGGAAGGAAAAACGATAAAAGCACAAAATGTGACATACCACGTCTAGGTGAAATTCTACTAATTATCTTATCTTTTACTTACACTTAAGCTTACGTCTGTGCTGG	PMR1 KO repair oligonucleotide
11292	TCGCCCCGTTCTTTCCATTC	PMR1 mutant allele amplification
11293	GGGCGAAAAGGTAAGAACGC	PMR1 mutant allele amplification
13522	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCCATAAAAGAACATTGCACCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for FRE2 gRNA plasmid
13539	AATAAAGTCTTTTTTATCCAAAGCTTATGAAACCCAACGAATATAAGTAGAATTTCACCTAGACGTGGTCATTTTTTACTTAAAACTAGTCATTTCATTAATAATACCTATCC	FRE2 KO repair oligonucleotide
13540	GGATAGGTATTATTAATGAAATGACTAGTTTTAAGTAAAAAATGACCACGTCTAGGTGAAATTCTACTTATATTCGTTGGGTTTCATAAGCTTTGGATAAAAAAGACTTTATT	FRE2 KO repair oligonucleotide
13524	GATCCCCCTTTGTGCATTGC	FRE2 mutant allele amplification
13525	TGGCTCAATGATGCTAGTGGG	FRE2 mutant allele amplification
12174	GCATCGTCTCATCGGTCTCATATGTCTGCTACCTCTACTGCTACTTCC	THI4 with YTK part 3 compatible overhangs
12175	ATGCCGTCTCAGGTCTCAGGATCTAAGCAGCAAAGTGTTTCAAAATTTG	THI4 with YTK part 3 compatible overhangs
14586	ACAGTTTTGACAACTGGTTACTTCCCTAAGACTGTTTATATTAGGATTGTCAAGACACTCCAGTTCGAGTTTATCATTATCAATAC	THI4↑ cassette repair for integration
14587	ATAATTATAATATCCTGGACACTTTACTTATCTAGCGTATGTTATTACTCGATAAGTGCTCGTTCAGGGTAATATATTTTAACC	THI4↑ cassette repair for integration
13518	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCTGAATCTGGTGATAGCACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for GAL11 gRNA plasmid
13533	TACTCAAAGATCAAGGATTAAAACGCTATTTCTTTTAAATCTGCTAGTAGAATTTCACCTAGACGTGGACATTTGAAGTTTCCATACTTTTGATACTTTTGAAGTTACTTCGT	GAL11 KO repair oligonucleotide
13534	ACGAAGTAACTTCAAAAGTATCAAAAGTATGGAAACTTCAAATGTCCACGTCTAGGTGAAATTCTACTAGCAGATTTAAAAGAAATAGCGTTTTAATCCTTGATCTTTGAGTA	GAL11 KO repair oligo
13498	TTCGAATCGGGCCTTCCTTC	GAL11 mutant allele amplification
13499	TGCTTGAAGTGGCACTTTGC	GAL11 mutant allele amplification
13517	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCTGGAAGGGTAGACCATGACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for TUP1 gRNA plasmid
13531	TGATAAGCAGGGGAAGAAAGAAATCAGCTTTCCATCCAAACCAATAGTAGAATTTCACCTAGACGTGGGAACAGAACACAAAAGGAACACTTTACAAATGTAACTAACTAAAC	TUP1 KO repair oligonucleotide
13532	GTTTAGTTAGTTACATTTGTAAAGTGTTCCTTTTGTGTTCTGTTCCCACGTCTAGGTGAAATTCTACTATTGGTTTGGATGGAAAGCTGATTTCTTTCTTCCCCTGCTTATCA	TUP1 KO repair oligonucleotide
15077	CACGCCAAGTTACCTTTCGC	TUP1 mutant allele amplification
15078	GGAAGGGATGAATGGTGAGG	TUP1 mutant allele amplification
13519	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCGAAAAAGAGAAGGCAAAACGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for ISW2 gRNA plasmid
13535	CTTGTTGGTTTAAGTCGTAACAAAAGGAAAACTTACAATCAGATCAGTAGAATTTCACCTAGACGTGGATCATGTATTGTGCATTAAAATAAGTGACGTGAGAGATATAATTT	ISW2 KO repair oligonucleotide
13536	AAATTATATCTCTCACGTCACTTATTTTAATGCACAATACATGATCCACGTCTAGGTGAAATTCTACTGATCTGATTGTAAGTTTTCCTTTTGTTACGACTTAAACCAACAAG	ISW2 KO repair oligonucleotide
13496	TCACCCAGAGGCAAAAGGTG	ISW2 mutant allele amplification
13497	TAGTTAAAGCGGCTCGACCC	ISW2 mutant allele amplification
16598	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCTCAAGATTGTCTTGTTCTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC	2-μm fragment for FMS1 gRNA plasmid
13527	AACAAGAAGTGAGTTAATAAAGGCAAAAACAGTGGTCGTGTGAGAAGTAGAATTTCACCTAGACGTGGAATCTATTTTTTCGAAATTACTTACACTTTTGACGGCTAGAAAAG	FMS1 KO repair oligonucleotide
13528	CTTTTCTAGCCGTCAAAAGTGTAAGTAATTTCGAAAAAATAGATTCCACGTCTAGGTGAAATTCTACTTCTCACACGACCACTGTTTTTGCCTTTATTAACTCACTTCTTGTT	FMS1 KO repair oligonucleotide
13525	TGGCTCAATGATGCTAGTGGG	FMS1 mutant allele amplification
13526	AGCCAAATTGCCAAGAAAGGG	FMS1 mutant allele amplification
16601	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCGCGTGAACGCAAATGCATCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC	2-μm fragment for SPE2 gRNA plasmid
16602	AATAGTATTTTTCAGCGAGAATCATATTGGATGAGTATCCACATGGCGTGAACGCAAATGtATCGTGaTGAAATGATAAATCGGAGTCTTGGGCCGAGTTGACATATATTTCGTCAAG	SPE2 mutation-carrying repair oligonucleotide
16603	CTTGACGAAATATATGTCAACTCGGCCCAAGACTCCGATTTATCATTTCAtCACGATaCATTTGCGTTCACGCCATGTGGATACTCATCCAATATGATTCTCGCTGAAAAATACTATT	SPE2 mutation-carrying repair oligonucleotide
12174	GCATCGTCTCATCGGTCTCATATGTCTGCTACCTCTACTGCTACTTCC	YTK-compatible end addition to THI4 CDS
12175	ATGCCGTCTCAGGTCTCAGGATCTAAGCAGCAAAGTGTTTCAAAATTTG	YTK-compatible end addition to THI4 CDS
12985	TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCAAACATTCAAATATATTCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG	2-μm fragment for YPRcTau3 gRNA plasmid
13261	AATACGAGGCGAATGTCTAGG	THI4 integration check
13262	GCCTCCCCTAGCTGAACAAC	THI4 integration check
13492	TACAGCTCGCTCCTTGCATC	SPE2 mutation check
13493	GCTTGCTTGGAGGGCTTTTC	SPE2 mutation check

^aKO, knockout.