Table 1.
Primer | Gene | Nucleotide sequencea | Use |
---|---|---|---|
Plasmids for gene disruption | |||
ndxA5-f | ndxA | 5′-GCGGCCGCCAAGTGCTGGACCTGTACGA-3′ | 5′ region of ndxA |
ndxA5-r | 5′-GCGGCCGCGGTATCGGGTGGAGATGAGA-3′ | ||
ndxA3-f | 5′-GAATTCACCCGCTACGATGTTACCTG-3′ | 3′ region of ndxA | |
ndxA3-r | 5′-CTCGAGAATATGGAAGCGTCCTCACG-3′ | ||
ndxB5-f | ndxB | 5′-GCGGCCGCGTTAGATCCGCTCACCGAAG-3′ | 5′ region of ndxB |
ndxB5-r | 5′-GCGGCCGCAAGCGAATCTCAAGCGACAT-3′ | ||
ndxB3-f | 5′-GAATTCCCGTAAGGACTGCCCATTTA-3′ | 3′ region of ndxB | |
ndxB3-r | 5′-CTCGAGCAAAGGTCAGCGCACATCTA-3′ | ||
ndxC5-f | ndxC | 5′-GCGGCCGCCTCGAAAGGGAATGAACCAA-3′ | 5′ region of ndxC |
ndxC5-r | 5′-GCGGCCGCTCTTGCCTGCAGAAAACTCA-3′ | ||
ndxC3-f | 5′-GAATTCCGTGGACCCAACTTCTGTATATC-3′ | 3′ region of ndxC | |
ndxC3-r | 5′-CTCGAGGAACTCCAACTGTGGTTGATAGG-3′ | ||
sirA5-f | sirA | 5′-GCGGCCGCGCTCGATCTCAAAACGGAAG-3′ | 5′ region of sirA |
sirA5-r | 5′-TCTAGATCTTCGACATTGCTGTCGTC-3′ | ||
sirA3-f | 5′-GAATTCATCCTTGCCCAACAGATCAC-3′ | 3′ region of sirA | |
sirA3-r | 5′-CTCGAGGGAAGCTCCGTCCATCAATA-3′ | ||
ndxA3-f2 | ndxA | 5′-CTGCAGACCCGCTACGATGTTACCTG-3′ | 3′ region of ndxA (for insertion into pPyrG plasmid) |
ndxA3-r2 | 5′-GAATTCAATATGGAAGCGTCCTCACG-3′ | ||
Plasmids for GFP-fused protein production | |||
gpdpro-f | gpdA | 5′-CCAATGCATTGGAAAAGTCACACAACACAAGC-3′ | gpdA gene promoter |
gpdpro-r | 5′-GCGGCCGCTGTGATGTCTGCTCAAGC-3′ | GFP-NdxC fusion | |
gfpN-f | gfp | 5′-GCGGCCGCATGGTGAGCAAGGGCGAGGAGCTG-3′ | |
gfpN-r | 5′-TCTAGACTTGTACAGCTCGTCCAT-3′ | NdxA- or NdxB-GFP fusion | |
gfpC-f | gfp | 5′-TCTAGAATGGTGAGCAAGGGCGAGGAGCTG-3′ | NdxA-GFP fusion |
gfpC-r | 5′-GGATCCTTACTTGTACAGCTCGTCCAT-3′ | ||
gndxA-f | ndxA | 5′-GCGGCCGCATGCCCACAGAGACAAAATCCGTTCGCGTT-3′ | NdxB-GFP fusion |
gndxA-r | 5′-TCTAGAGTTCAAAACAGGCTGAAAA-3′ | ||
gndxB-f | ndxB | 5′-GCGGCCGCATGTCAGATCCAATGTACGCCAGGTCAAA-3′ | GFP-NdxC fusion |
gndxB-r | 5′-TCTAGAGAGCTTCAGTTTCTTCGCC-3′ | ||
gndxC-f | ndxC | 5′-TCTAGAATGTCCAATAATTCACAAATCC-3′ | DsRed-SKM |
gndxC-r | 5′-GGATCCCTACATCTTCGACTTGTCTGCAAG-3′ | ||
ds-red-f | ds-red | 5′-GCGGCCGCATGGCCTCCTCCGAGGACGT-3′ | |
ds-red-r | 5′-GGATCCCTACATCTTCGACAGGAACAGGTGGTGGCGG-3′ | ||
Plasmids for recombinant protein production | |||
rndxA-f | ndxA | 5′-CATATGCCGACCGAAACCAAATCCGTTCGCGTT-3′ | pETndxA |
rndxA-r | 5′-AAGCTTTCAGTTCAAAACAGGCTGAAAA-3′ | ||
rndxB-f | ndxB | 5′-CATATGTCAGATCCGATGTACGCCCGCTCAAA-3′ | pETndxB |
rndxB-r | 5′-AAGCTTTCAGAGCTTCAGTTTCTTCGCC-3′ | ||
rndxC-f | ndxC | 5′-CATATGTCCAATAATTCACAAATCC-3′ | pETndxC |
rndxC-r | 5′-AAGCTTCTACATCTTCGACTTGTCTGCAAG-3′ | ||
rndxD-f | ndxD | 5′-GGATCCGTGGACCAGG TTCGTTCAAT-3′ | pETndxD |
rndxD-r | 5′-GCGGCCGCCTATCTCTTCATGGAACTTC-3′ | ||
rSirA-f | sirA | 5′-CGGGATCCATGGACCTTGCTTCAGCGCCCCGTGGGGAGA-3′ | pETsirA |
rSirA-r | 5′-GCGGCCGCTCCGCTAACCTTGAATACATGTCGTGA-3′ | ||
Site-directed mutagenesis | |||
E57Q-f | ndxA | 5′-TGCGCGGCTCGCCAATTAATAGAAGAA-3′ | pETndxA-E57Q |
E57Q-r | 5′-TTCTTCTATTAATTGGCGAGCCGCGCA-3′ | ||
E61Q-f | ndxA | 5′-GAATTAATAGAACAAACGGGGGTACAT-3′ | pETndxA-E61Q |
E61Q-r | 5′-ATGTACCCCCGTTTGTTCTATTAATTC-3′ | ||
H286N-f | sirA | 5′-ATTGTACAGTGCAACGGCTCTTTTGCC-3′ | pETsirA-H286N |
H286N-r | 5′-GGCAAAAGAGCCGTTGCACTGTACAAT-3′ | ||
Plasmids for expressing ndxA and sirA in A. nidulans | |||
ndxA-f | ndxA | 5′-GCGGCCGCTGACAGGGTACCAATGCAAA-3′ | pBSndxA, pBSndxA-E57Q |
ndxA-r | 5′-GCGGCCGCAGCTCGCAGTTTTGTCCAAT-3′ | pBSsirA, pBSsirA-H286N | |
sirA-f | sirA | 5′-GCGGCCGCTCTCCCGAGCCTCTTATCAA-3′ | |
sirA-r | 5′-GCGGCCGCTTCAGTATAGCCCCGCACTC-3′ | ||
Quantitative PCR | |||
RTactA-f | actA | 5′-GAAGTCCTACGAACTGCCTGATG-3′ | |
RTactA-r | 5′-AAGAACGCTGGGCTGGAA-3′ | ||
RTndxA-f | ndxA | 5′-GGACGGGAAGCACTACGTTA-3′ | |
RTndxA-r | 5′-CAGCTTCGACCTGCTTTTTC-3′ | ||
RTndxB-f | ndxB | 5′-CGTGAGCTCAAGGAGGAGAC-3′ | |
RTndxB-r | 5′-AGTTGTGGCTTGGGATTTTG-3′ | ||
RTndxC-f | ndxC | 5′-AGCAACAACACAATGCGAAG-3′ | |
RTndxC-r | 5′-AGTAGCTGTTGCGGGTGTTC-3′ | ||
RTaflR-f | aflR | 5′-CTGCCTTGCGAGTATATGGTTTC-3′ | |
RTaflR-r | 5′-TTGGTGATGGTGCTGTCTTG-3′ | ||
RTstcU-f | stcU | 5′-CATTTCCATTCAAGCCGATGT-3′ | |
RTstcU-r | 5′-CCAGGTATCCGAAGTGCTCAA-3′ | ||
RTipnA-f | ipnA | 5′-CAGCGTGATTGATCCATTTG-3′ | |
RTipnA-r | 5′-CTAAATCCAACTCCCGTCCA-3′ | ||
RTpenD-f | penDE | 5′-AATGGGCGGATAGTGCATAC-3′ | |
RTpenD-r | 5′-CCGTCAAAACCAGAGAGGAG-3′ | ||
RTsirA-f | sirA | 5′-CGCATTCAGGAATCAAACCT-3′ | |
RTsirA-r | 5′-GCGTGTTCCCTGAAAACATT-3′ | ||
ChIP assay | |||
ipnA #1-f | ipnA #1 | 5′-TAATCCACGCAATCCACTGA-3′ | 5′ region of ipnA |
ipnA #1-r | 5′-CCGGTGCATAATGACAAGTG-3′ | ||
ipnA #2-f | ipnA #2 | 5′-TGAGAGCTACGCTTCCCATT-3′ | 5′ region of ipnA |
ipnA #2-r | 5′-AGTTCTCCAAAGCTGGCTCA-3′ | ||
ipnA #3-f | ipnA #3 | 5′-CGTGCCTACAACAAAGAGCA-3′ | ipnA |
ipnA #3-r | 5′-AGTTGTGGCTTGGGATTTTG-3′ | ||
aflR #4-f | aflR #4 | 5′-GACTCCTAGACCCCGACAGG-3′ | 5′ region of aflR |
aflR #4-r | 5′-TACTGCGGGCTAGAACTGGT-3′ | ||
aflR #5-f | aflR #5 | 5′-ATCTCCTCATGGCGAATCTC-3′ | 5′ region of aflR |
aflR #5-r | 5′-TATTCCCGCAGGGATTACAG-3′ | ||
aflR #6-f | aflR #6 | 5′-GCTCCAGATCCAAGGTCAAG-3′ | aflR |
aflR #6-r | 5′-CGTATTCGTCGGTGTTGTTG-3′ | ||
actApro-f | actA | 5′-TACTCCGCCTACCGCTACAA-3′ | 5′ region of actA |
actApro-r | 5′-GGAAGGGAGGAGGAGAGATG-3′ | ||
Producing SirA-HA protein | |||
SirAHA-f | sirA-HA | 5′-ATAAGAATGCGGCCGCATGGACCTTGCTTCAGCGCCCCGT-3′ | pSirA-HA |
SirAHA-r | 5′-CTCGAGCTACGCATAGTCCGGGACGTCATAGGGATAGGATCCCGCATAGTCAGGAACATCGTATGGGTAATAGCCCGCATAGTCAGGAACATCGTATGGGTATCCGCTAACCTTGAATACATGTCGTGA-3′ |
Restriction sites are underlined. Silent mutations are double underlined.