. 2019 Nov 13;4(6):e00610-19. doi: 10.1128/mSphere.00610-19

TABLE 2.

Oligonucleotide primers used in strain construction

Primer	Description	Sequence (5′ to 3′)^a
P118	Long homologous tail knockout primer KIP1::HIS1/LEU2/ARG4 5′	GTTGTTGTTGTTTTCATTCTTCATCTTGTGATTTCAGTTAAATTAATACTCATA GCAGCATTATCATCAACCAGTGTGATGGATATCTGC
P119	Long homologous tail knockout primer KIP1::HIS1/LEU2/ARG4 3′	AAATAAACCTCACAATTAATTAAACATGTACTGAACAAATGGAGTAAAACA AATATTGGTCTAATTATAAGCTCGGATCCACTAGTAACG
P120	−500 bp KIP1 check 5′	CGCACAAGACCTGGCACAAGAGAA
P121	+500 bp KIP1 check 3′	ATGGGCCAATGGGATCACATGG
P11	HIS1 check right 3′	AACACAACTGCACAATCTGGC
P12	HIS1 check left 5′	ATTAGATACGTTGGTGGTTCAGTT
P13	LEU2 check left 3′	AGAATTCCCAACTTTGTCTGTTC
P14	LEU2 check right 5′	AAACTTTGAACCCGGCTGCG
P247	KIP1 gRNA for fragment B stitching	CGTAAACTATTTTTAATTTGCGAAGTAATACTGCTTGTGGGTTTTAGAGCTAG AAATAGC
P248	KIP1 donor DNA with mini-AT 5′	ATTCTTCATCTTGTGATTTCAGTTAAATTAATACTCATAGCAGCggGACCAATA TTTGTTTTACTCCATTTGTTCAGTACATGTTTAATTAATTGT
P249	KIP1 donor DNA with mini-AT 3′	ACAATTAATTAAACATGTACTGAACAAATGGAGTAAAACAAATATTGGTCccG CTGCTATGAGTATTAATTTAACTGAAATCACAAGATGAAGAAT
P240	KIP1 tetO-SAT^R flipper 5′	ATTCATTCATTCAATCAGAGTAGTTTTAATATCTTCTTATAGTGGCCTGCATAT AGTTCAATCACGACGGAAACAGCTATGACCATG
P241	KIP1 tetO-SAT^R flipper 3′	GAGATTTAGCAGCAATCTCTTGAGAGTTCCTTCCTCGACATCTAACAACAAC TTGGATATTTGACCGCGGCGACTATTTATATTTGTATG
P242	tetO check 3′	AGTTATTGAATCTATTACTCAATCG
P170	KIP1 ORF confirmation primer 3′	CTTCATTCACTATATTTCCAACTTGTGATTG
P128	KIP1 into pCIp10 (MluI) primer 5′	GGACCGACGCGTCACAGAGAGAGAGAGAGAGAGAAAGAGAATGAG
P129	KIP1 into pCIp10 (KpnI) primer 3′	GGACCGGGTACCCATCATCAACATAATCAACCACATCACCCACA
P199	Long homologous tail knockout primer KAR3::HIS1/LEU2/ARG4 5′	TCAAAAAGTTGCCAGACAGGTTTTTTACAATTTTGAAACTACAATCCAATAG TCAATCGTGCACAAGTAACCAGTGTGATGGATATCTGC
P200	Long homologous tail knockout primer KAR3::HIS1⁺/LEU2⁺/ARG4⁺ 3′	TATATCTGAGCCAATATTTAAATAGATTCTTGTATATAAGTCATGTATGTAAAC TATTAACGTAGTAATAGCTCGGATCCACTAGTAACG
P201	−1,000 bp KAR3 check 5′	GTCCCAACTTCTCCTTATTGACTTCTT
P202	+1,000 bp KAR3 check 3′	GTTGCCTAAAATTCCTAAGGACCT
P212	ARG4-MAL2-KAR3 long homologous primer 5′	AAAGAAAAACTTGCCCATCTCATCGAGAGTCTAATTTCTTACGCGGGAACTAG AAAAAAAAAACTGAAGAAGCTTCGTACGCTGCAGGTC
P213	ARG4-MAL2-KAR3 long homologous primer 3′	CCACCTAAAAGATTTGATGGTTGTGACACATTTAGAAATTTATGTTTAGTATTT TCGTCACTCATTGTAGTTGATTATTAGTTAAACCAC
P16	ARG4 check left 3′	TTCCATTTAGAGAAACTCATCATATTT
P17	SAT1^R check left 3′	CATACCACCGTCCATTTTGAATG
P18	SAT1^R check right 5′	TGATGAAGACTCTGCTTGCTATG
P137	KIP1-GFP-ARG4 or SAT1^R long-tailed primer (C terminal) 5′	TTCTACCACGACCAATAATAATAAAAAGAGAAAAATATTACAAACAATGGAC AATTTATTAGGTGGTGGTTCTAAAGGTGAAGAATTATT
P187	KIP1-GFP-ARG4 or SAT1^R long-tailed primer (C terminal) 3′	CATATATTATATATTAATATTATTAAGAGTTTTTGGAAATATGGAACTATAAT GAGGAGGACCACCTTTGATTGTAAATAGTAATAATTA
P69	GFP sequencing/left junction check 3′	GATCTGGGTATCTAGCAAAAC
P169	KIP1 ORF confirmation primer 5′	GCACAAGTCAATCTACTGGAAACAT
P284	KIP1-mScarlet localization long-tail 5′	TGTTGTTGTTTTCATTCTTCATCTTGTGATTTCAGTTAAATTAATACTCATAGC AGCATTATCATCAGACTCACTATAGGGCGAATTGGG
P285	KIP1-mScarlet localization long-tail 3′	AAATAAACCTCACAATTAATTAAACATGTACTGAACAAATGGAGTAAAACA AATATTGGTCTAATTATACAAAAGCTGGAGCTCCACCGC
P254	mScarlet check 5′	GTAGATATTTGGCTGATTTCAAAAC
P108	SPC98-GFP-SAT1^R long-tailed primer for pGFP-SAT1 5′	TTTGAAAAATGATTTGAATAGAGATTATAATTTAAAGGATCTTAGTAAGTTGTT GGTGGTGGTTCTAAAGGTGAAGAATTATTCACTGG
P109	SPC98-GFP-SAT1^R long-tailed primer for pGFP-SAT1 3′	TGAGCTTTACAGAGATCTTGTCGGTAATCATAGATTTCCCCACTTGTTCTGTAA TCGACGAAATTGAGGACCACCTTTGATTGTAAATAG
P110	SPC98-GFP integration check 3′	GCAGCGTCCACCCTTTGTAAAAGTG
P107	pGAL1-Tub2-GFP/mCherry/mNeon downstream check 3′	TATTATCTATATTGTCAAGCCAAGACAAGCCCATT
P243	NOP1-mScarlet long-tail 5′	ACCTTATGAAAGAGACCATTGTATTGTTGTTGGTAGATACATGAGAAGCGGA ATAAAGAAAGGTGGTAGTGGTATGGTTTCTAAAG
P244	NOP1-mScarlet long-tail 3′	AAGGTCAAAGTGCCATCAAAGGTGTGTTATTGGGTTCATTATCAAATTATTTG GTGACAAGGCGGCCGCTCTAGAACTAGTGGATC
P246	NOP1 check 3′	CGATTGAACATGTTAAACAAAGC

The portion of primer homologous to plasmid template is in boldface and italic. The restriction enzyme cut site is underlined. Lowercase letters represent the mini-ADD-TAG sequence (mAT [gg]) for subsequent CRISPR-mediated gene editing (89).