Supplementary Table S3
A. Linker Scanning Primers
Primer Name / Primer sequence / Location of mutationYHB1_5’_F / GCGGTACCTTCACTACAGAATTACGATCG
YHB1-ORF-R / GCCTGCAGAGGATCGTAAGGACCAAAATATTCA
Msub1-R1 / GGCTCGAGCCCTTGTCTCATTCTCTCGGTCTTTAAACT / -481 to -472
Msub1-F2 / GGCTCGAGCCTGAGAACTAAAAAAAACCCAAATGGAG
Msub2-R1 / GGCTCGAGACTTTTTTTTAACTTGTCTCATTCTCTCGGTC / -471 to -462
Msub2-F2 / GTCTCGAGCCAAAAAACCCAAATGGAGTATTTAGGT
Msub3-R1 / GTCTCGAGGGTTAGTTCTCATTTTTTTTAACTTGTCTCATTCTC / -461 to -452
Msub3-F2 / CCCTCGAGACAATGGAGTATTTAGGTTAAAGCGCTC
Msub4-R1 / CGCTCGAGGGTGGGTTTTTTTTAGTTCTC / -451 to -442
Msub4-F2 / CCCTCGAGCGTTAGGTTAAAGCGCTCCGGCATT
Msub5-R1 / TACTCGAGTATTTAACCTAAATACTCCATTTGGGTT / -441 to -432
Msub5-F2 / GGCTCGAGCCGCGCTCCGGCATTTCCCAACT
Msub6-R1 / TACTCGAGTATTTAACCTAAATACTCCATTTGGGTT / -431 to -422
Msub6-F2 / TACTCGAGTAATTTCCCAACTCCTCCATCATTCA
Msub6-R1a / AAAATTACTCGAGTATTTAACCTAAATACTCCATTTGGG / -431 to -422
Msub6-F2a / TTAAATACTCGAGTAATTTTCCCAACTCCTCCATCATTC
Msub7-R1 / GGCTCGAGCGGCCGGAGCGCTTTAACCTAAATA / -421 to -412
Msub7-F2 / CGCTCGAGCCCTCCTCCATCATTCATTCATTCTTCTTC
Msub8-R1 / TCCTCGAGCTTTGGGAAAATGCCGGAGC / -411 to -402
Msub8-F2 / AGCTCGAGGAATTCATTCATTCTTCTTCTTTAAAT
Msub9-R1 / CGCTCGAGCGGATGGAGGAGTTGGGAAAATGC / -401 to -392
Msub9-F2 / CGCTCGAGCGTCTTCTTCTTTAAATTGAATATTACAC
Msub10-R1 / CCCTCGAGTCATGAATGAATGATGGAGGAGTTGGG / -391 to -382
Msub10-F2 / GACTCGAGGGTAAATTGAATATTACACAAAACCGCGGA
Msub11-R1 / CGCTCGAGGCAAGAAGAAGAATGAATGAATGATGGAGG / -381 to -372
Msub11-F2 / GCCTCGAGCGATTACACAAAACCGCGGAAAAAAC
Msub12-R1 / GGCTCGAGCGATTCAATTTAAAGAAGAAGAATGAATG / -371 to -362
Msub12-F2 / CGCTCGAGCCACCGCGGAAAAAACACCTGAT
Msub13-R1 / GGCTCGAGTGTTTGTGTAATATTCAATTTAAAGAAG / -361 to -352
Msub13-F2 / CACTCGAGCCAAACACCTGATGATTTACTCAAAACA
Msub14-R1 / AAATAGTCGCTCGAGTTCATGTAATAATTGAAACAACATCTCC
Msub14-F2 / ATGAACTCGAGCGACTATTTAAAACCATTAAACATGTTTTTTTC
Msub15-R1 / GTTTTAACTCGAGGTCTCCCCGACGGTAATAATTG
Msub15-F2 / GGGAGACCTCGAGTTAAAACCATTAAACATGTTTTTTTC
Msub16-R1 / CGTACCGTCTCGAGCACTGTTTAATGGTTTTCCCGCTG
Msub16-F2 / AAACAGTGCTCGAGACGGTACGTTTGTTTTCCTTTTTTT
Msub17-R1 / ATTACATCTCGAGCTCATCAGCGGGAAAACCATTAAAC
Msub17-F2 / ATTACATCTCGAGCTCATCAGCGGGAAAACCATTAAAC
Msub18-R1 / CCCGACGCTCGAGATTGAAACAACATCTCCAATTTAC
Msub18-F2 / TTTCAATCTCGAGCGTCGGGGAGCATCAGCGGG
Msub19-R1 / CTCCCCGCTCGAGATAATTGAAACAACATCTCCAATT
Msub19-F2 / CAATTATCTCGAGCGGGGAGCATCAGCGGGAAAA
Msub20-R1 / TAATACGCTCGAGCCCATCTCCAATTTACACTTATAC
Msub20-F2 / AGATGGGCTCGAGCGTATTACCGTCGGGGAGCATC
Msub21-R1 / AACAAACCTCGAGCGTTACACTTATACAGAAAATG
Msub21-F2 / TGTAACGCTCGAGGTTTGTTTCAATTATTACCGTC
Msub22-R1 / CTGATGCTCGAGGACGGTAATAATTGAAACAAC
Msub22-F2 / ATTACCGTCCTCGAGCATCAGCGGGAAAACCA
Msub23-R1 / AAATAGTCGTTTAAACTCATGTAATAATTGAAACAACATCTCC
Msub23-F2 / ATGAGTTTAAACGACTATTTAAAACCATTAAACATGTTTTTTTC
Msub24-R1 / CCGACGGCTCGAGTTGAAACAACATCTCCAATTTAC
Msub24-F2 / GTTTCAACTCGAGCCGTCGGGGAGCATCAGCGG
Msub25-R1 / CCGCTGACTCGAGCCGACGGTAATAATTGAAACAAC
Msub25-F2 / CCGTCGGCTCGAGTCAGCGGGAAAACCATTAAAC
Msub26-R1 / CGACGGTCTCGAGTGAAACAACATCTCCAATTTAC
Msub26-F2 / TGTTTCACTCGAGACCGTCGGGGAGCATCAGC
Msub27-R1 / GACGGTACTCGAGGAAACAACATCTCCAATTTACAC
Msub27-F2 / TTGTTTCCTCGAGTACCGTCGGGGAGCATCAG
Msub28-R1 / CGCTGATCTCGAGCGACGGTAATAATTGAAACAAC
Msub28-F2 / ACCGTCGCTCGAGATCAGCGGGAAAACCATTAAAC
Msub29-R1 / ACGGTAACTCGAGAAACAACATCTCCAATTTAC
Msub29-F2 / GTTGTTTCTCGAGTTACCGTCGGGGAGCATCAG
Msub30-R1 / CGGTAATCTCGAGAACAACATCTCCAATTTACAC
Msub30-F2 / TGTTGTTCTCGAGATTACCGTCGGGGAGCATCAG
Msub31-R1 / ACGGTAACAATTGAAACAACATCTCCAATTTAC
Msub31-F2 / GTTGTTTCAATTGTTACCGTCGGGGAGCATCAG
Checking primers:
Msub border F1 / CTGGTGCCTATCTTGGTCGATATGAAA
Msub border R1 / GATTCATGGTTATATCTTTATAGAAATCGAAGG
Msub border F2 / GGTACTGAAACTAAACCAGCTGCTCCAAA
Msub border R2 / CCTGGAGTTGGATTAGATGATAAAGGTGA
Construction of mutant regulatory regions
à PCR 1: Primer pair YHB1_5’_F + Msub(#) R1
à PCR 2: Primer pair YHB1-ORF-R + Msub(#) F2
à PCR 3: Fusion - YHB1_5’_F + YHB1-ORF-R.
Confirmation of correct location of plasmid insertion
à PCR 1: Msub border F1 + Msub border R1
à PCR 2: Msub border F2 + Msub border R2
B. DNA oligomers used in isolating NORE-binding proteins
wtNORE33F-biotin / biotin-TGTTGTTTCAATTATTACCGTCGGGGAGCATCA
wtNORE33R / TGATGCTCCCCGACGGTAATAATTGAAACAACA
Msub19NORE33F-biotin / biotin-TGTTGTTTCAATTATCTCGAGCGGGGAGCATCA
Msub19NORE33F / TGTTGTTTCAATTATCTCGAGCGGGGAGCATCA
Msub19NORE33R / TGATGCTCCCCGCTCGAGATAATTGAAACAACA
C. Gene deletion primers
Universal primer 2 / ccgctgctaggcgcgccgtgACCAGTGTGATGGATATCTGC
Universal primer 5 / gcagggatgcggccgctgacAGCTCGGATCCACTAGTAACG
ZCF36 deleterF 1 / AAACTTTGCGGGAGGAATTG
ZCF36 deleterR 3 / cacggcgcgcctagcagcggCAAGAAAAAAAAAGAAGGGCA
ZCF36 deleterF 4 / gtcagcggccgcatccctgcTGGTGAATTGGCAGCTCTTA
ZCF36 deleterR 6 / GGGCTCATTACTGGCTTTTT
STB5 deleterF 1 / CGATCTCTTCTTTCGAAAAGG
STB5 deleterR 3 / cacggcgcgcctagcagcggTGAGCAAAGGTTGTAGGGAA
STB5 deleterF 4 / gtcagcggccgcatccctgcAAATGCCTTGATTGTGTGCC
STB5 deleterR 6 / TTCAAGGGATGTGCTACATGG
CTA4 deleterF 1 / CTCCGTTTCCAAAAAGAAAAA
CTA4 deleterR 3 / cacggcgcgcctagcagcggCAAGTATTCCAAATGGTTCCG
CTA4 deleterF 4 / gtcagcggccgcatccctgcTGAATCACTTGTAATTTCCAACC
CTA4 deleterR 6 / CAGGTACTAAGAATGACAAGCAGA
ZCF29 deleterF 1 / TCATCCCTCAAACCAAGTCA
ZCF29 deleterR 3 / cacggcgcgcctagcagcggTCGAATTTGCGTGTTCTTCG
ZCF29 deleterF 4 / gtcagcggccgcatccctgcATTCTGTTTGGGAGGAGGTCTT
ZCF29 deleterR 6 / TTCTGTCACATCTTTTGACTTTGG
WAR1 deleterF 1 / CTGAATACTTGACTTCTGTTTTCA
WAR1 deleterR 3 / cacggcgcgcctagcagcggTGAAAGAACTTTGTTTGGAGG
WAR1 deleterF 4 / gtcagcggccgcatccctgcTGCCAAGAGTCAAGAAAGGAAAG
WAR1 deleterR 6 / GTCTCCATACTCCGAAAAATGAC
YHB1 deleterF 1 / AAATTGAATATTACACAAAACCG
YHB1 deleterR 3 / cacggcgcgcctagcagcggGCTGCTCTGTTCTAAAGTTGTCT
YHB1 deleterF 4 / gtcagcggccgcatccctgcTGTGGGGGGTGTATATCTTTAGA
YHB1 deleterR 6 / TTAATGAACTTGGTGTGATGATGT
CaLEU2 primer 1 / AAGCCGAAGTCGACTATGTC
CaLEU2 primer 3 / cacggcgcgcctagcagcggGGATATTGGTTTTAAAAGAAAGG
CaLEU2 primer 4 / gtcagcggccgcatccctgcACAGTATATACAGTAGTTAGC
CaLEU2 primer 6 / TGACAAATGAATTCAGTCAG
CaARG4 primer 1 / GGACAGAAAGTTATTGTACAG
CaARG4 primer 3 / cacggcgcgcctagcagcggGATTATTCTTGATAGCTGTTATG
CaARG4 primer 4 / gtcagcggccgcatccctgcGTCATATAATAATCACAGTATTG
CaARG4 primer 6 / TCAGACGATCTTTACAATGG
SSU1 deletion upstream 5’ / GTACGGTACCCACCAAGGTACTTTCATGAAC
SSU1 deletion upstream 3’ / GCGTCGTATGTGACTAGACTGGATCCCTCGAAGACGACATTATTG
SSU1 deletion downstream 5’ / GGATCCAGTCTAGTCACATACGACGCGAAAGCATGGTCTAATAGG
SSU1 deletion downstream 3’ / ATGCCCGCGGGCACTTAATTTAGGCTAG
Insertion check primers / Primer Sequence
ARG4 5'check-R (+131) / GTACCAGTTAAATCTGCATCATACATAAC
ARG4 3'check-F (+1261) / GATTCACGTTTTGAAAAAGATGTTATGG
STB5 5' del check / AGTGGGGTATTTCTTAGAATTGGCAG
STB5 3' del check / GCCAATATACGAGCCTTGATTCGTT
CTA4 5' del check / CAGCATTCTAGTCATCTGACAACGT
CTA4 3' del check / GAAACTAAAGGGACAAGCATTTATCACA
ZCF36 5' del check / TGCAAATTTTAATCCAAACGCTGAC
ZCF36 3' del check / AGTATTGCCGAGCCCTGTTTATCA
WAR1 5' del check / TTCTAATGCCCTCATCATCCCTTG
WAR1 3' del check / TGATGGTTGATTTAAGGATCGCGT
ZCF29 5' del check / GCTTTGTTCTTATAGTATCGCCAC
ZCF29 3' del check / TCACCACCAACAAACAGAACAAGG
YHB1 5' del check / GGAGTATTTAGGTTAAAGCGCTC
YHB1 3' del check / GATGTAAGGGTTGATGATGCCAAGT
LEU2 5' del check / CTTCGGCATCTTTGGAACCATCATA
LEU2 3' del check / GACCAGACTCTATCCCAAAG
ARG4 5' del check / CAAGAGTAGTCTCAAATAAACC
ARG4 3' del check / CGTTTGGAAGCTGTATATCG
SAT1 5’ check for SSU1 / CTAGACCTCAAGTCTCGAACGAAACAGC
SSU1 3’ del check / TGCAGAGCTCGTCGACACCATTTCAGATTATAC
NOREBP intracheck primers / Primer Sequence
CTA4 intracheck-F / GCGTCACAAACCTGAAACCATTGAT
CTA4 intracheck-R / GGACAAGCATTGGGATCGTCACTATTA
STB5 intracheck-F / GAGTACCCCACATTGTTTGAATTGTGA
STB5 intracheck-R / CCTCAGCACATTTCTTTGCAACTATCC
WAR1 intracheck-F / AACCCCTCAAATCTAATGAATCTGACAA
WAR1 intracheck-Ra / ACGACAATACATCCAATGGTTCAGCAC
ZCF29 intracheck-F / GAAGTCCCCTTGTGTCTATTCAGAACAAC
ZCF29 intracheck-R / CCAGCATTATTAGTGTAATTGACGTGACCC
ZCF36 intracheck-F / GTCGAAAAAGAAAGATAAGATGTGATCGTC
ZCF36 intracheck-R / CTTTGTCCACCAAATCCATCAAAATTC
YHB1 intracheck-F / TCTATCAACGTATGATTGGAAATTATGATGAAG
YHB1 intracheck-Ra / GTCTTCAGAAAAGTATTCAATGGCTTGGA
Primer combinations:
Deletion of first allele
à PCR 1: (gene) deleterF 1 + (gene) deleterR 3, SN152 genomic DNA template
à PCR 2: (gene) deleterF 4 + (gene) deleterF 6
à PCR 3: Universal primer 2 + Universal primer 5 on plasmid pSN40 as template DNA
à PCR 4: fusion -(gene) deleterF 1 + (gene) deleterF 6 on products of PCRs 1, 2, and 3.
Insertion check for first allele
à PCR 1: (gene) 5’ del check + Universal primer 5
à PCR 2: Universal primer 2 + (gene) 3' del check
Deletion of second allele
à PCR 1: (gene) deleterF 1 + (gene) deleterR 3
à PCR 2: (gene) deleterF 4 + (gene) deleterF 6
à PCR 3: Universal primer 2 + Universal primer 5 on plasmid pSN69 as template DNA
à PCR 4: fusion -(gene)deleterF 1 + (gene) deleterF 6 on products of PCRs 1, 2, and 3.
Insertion check for second allele
(CdARG4 is oriented in the opposite direction in plasmid pSN69 that CmLEU2 was on plasmid pSN40)
à PCR 1: (gene) 5' del check + ARG4 3'check-F (+1261)
à PCR 2: ARG4 5'check-R (+131) + (gene) 3' del check
Confirmation that all gene copies are removed from genome
à (gene)intracheck-F + (gene)intracheck-R
Creating SLA1.1 isogenic wild type strain
creating CmLEU2 construct for CaLEU2 site
à PCR 1: CaLEU2 primer 1+ CaLEU2 primer 3
à PCR 2: CaLEU2 primer 4 + CaLEU2 primer 6
à PCR 3: Universal primer 2 + Universal primer 5 on plasmid pSN40 as template DNA
à PCR 4: fusion - CaLEU2 primer 1 + Universal primer 5 on products of PCRs 1 and 3.
à PCR 5: fusion - CaLEU2 primer 1 + CaLEU2 primer 6 on products of PCRs 2 and 4.
à
checking insertion
à PCR 1: LEU2 5' del check + Universal primer 5
à PCR 2: Universal primer 2 + LEU2 3' del check
creating CdARG4 construct for CaARG4 site
à PCR 1: CaARG4 primer 1 + CaARG4 primer 3, SN152 genomic DNA template
à PCR 2: CaARG4 primer 4 + CaARG4 primer 6
à PCR 3: Universal primer 2 + Universal primer 5 on plasmid pSN40 as template DNA
à PCR 4: fusion - CaARG4 primer 1 + Universal primer 5 on products of PCRs 1 and 3.
à PCR 5: fusion - Universal primer 2 + CaARG4 primer 6 on products of PCRs 2 and 3.
à PCR 6: fusion - CaARG4 primer 1 + CaARG4 primer 6 on products of PCRs 4 and 5.
checking insertion
à PCR 1: ARG4 5' del check + ARG4 3'check-F (+1261)
à PCR 2: ARG4 5'check-R (+131) + ARG4 3' del check
D. DNA oligos used in Northern blot
TEF1 – 5’: ATAGTCATAATCAATCATGGGT
TEF1 – 3’: CTTACATAATATTCAACTAGC
YHB1 intracheck-F: TCTATCAACGTATGATTGGAAATTATGATGAAG
YHB1 intracheck-Ra: GTCTTCAGAAAAGTATTCAATGGCTTGGA
E. DNA oligos used for RT-PCR
SSU1 – 5’: ATCGGATGTGGAAGCCAAACTAAATG
SSU1 – 3’: AGAAACCCACAGGAAGGAAACTTGTC
ACT1 – 5’: GACCGAAGCTCCAATGAATCC
ACT1 – 3’: TCTCTTTCAGCACTAGTAGTG
F. Complementation Primers
CTA4 gene UP1 / TTAGAGCTCACCAACCATAAAACCTATTTCACG
CTA4 gene DN1 / TTAGCTAGCTGGATTTGATAATGTGAGATTGGTC
YHB1 gene UP1 / TTAGAGCTCACTACAGAATTACGATCGTTTCCCA
YHB1 gene DN1 / TTAGCTAGCATTCTGACAATTCTCTTTGTCTAGAGAGT
SSU1 gene UP1 / GTACGGTACCCACCAAGGTACTTTCATGAAC
SSU1 gene DN1 / TGACACGCGTCTCAATAGAAACTTCTACCAG
Primers to check insertion of complementation plasmid CIp20
CIp20-upcheck-F / GTAATAGTGTGTGTGTTCCAAGTCCCAGC
CIp20-upcheck-R / CCTATTGCTTTTTGACTATACCTTCGCTG
CIp20-downcheck-F / TTTATGCTTCCGGCTCGTATGTTGT
CIp20-downcheck-R / CGCCAAAGAGTTTCCCCTATTATCACTAC
Primer combinations:
Amplification of CTA4
à CTA4 gene UP1 + CTA4 gene DN1
Amplification of YHB1
à YHB1 gene UP1 + YHB1 gene DN1
Insertion check for first allele
à PCR 1: CIp20-upcheck-F + CIp20-upcheck-R
à PCR 2: CIp20-downcheck-F + CIp20-downcheck-R
G. Primers for construction of 9xMyc-HIS1 tagging plasmid pMGC2 and cassette for tagging CTA4
9xMyc-UP4 TAATAAGCTTCGGTTCTGCTGCTAGTGGTGAACA
9xMyc-DN2 TAATAGATCTTAGCTAGTGGATCCGTTCAAGTCTTC
CTA4-1: CGAGAATAATGCATCGGCAAT
CTA4-3: ccacggcgcgcctagcagcggTCCATCATCCATAAGGTCAAA
CTA4-4: gtcagcggccgcatccctgcTGAATCACTTGTAATTTCCAACC
CTA4-6: CAGGTACTAAGAATGACAAGCAGA
CTA4-1b GAGAATAATGCATCGGCAATAGTGACACCTTAT
CTA4-6b CAGGTACTAAGAATGACAAGCAGATAAAGAAAT
linking primer 2: ccgctgctaggcgcgccgtggGGTGACACTATAGAATACTCA
linking primer 5: gcagggatgcggccgctgacCCAGTGTGATGGATATCTGC
Primers for checking insertion:
linking primer 2: ccgctgctaggcgcgccgtggGGTGACACTATAGAATACTCA
CTA4 3' del check: GAAACTAAAGGGACAAGCATTTATCACA
CTA4-myc upcheck F: GTTGGTATGCTGCCCGATTTTGAAA
CTA4-myc upcheck R: CAAGAATACAAAACCAGATTTCCAGATTTCC
Primer combinations for checking insertion:
5' junction: CTA4 myc upcheck F + CTA4 myc upcheck R
3’ junction: linking primer 2 + CTA4 3' del check