Table 3.
Oligonucleotide primers used in this study
| Name | Sequence (5′→3′) |
|---|---|
| Plasmid/integration conformation | |
| A-tag amp fwd | AAATAAACAAATAGGGGTTCCGC |
| A-tag amp rev | GAAATGCTGGATGGGAAGCG |
| B-tag amp fwd | TCCCATATGATTGTCTCCGTAAGCTCG |
| B-tag amp rev | ACTCTGTCATATACATCTGCCGCAC |
| C-tag amp fwd | GCAAATGCCTGCAAATCG |
| C-tag amp rev | CGCGTGTACGCATGTAAC |
| D-tag amp fwd | GCTAAATGTACGGGCGACAG |
| D-tag amp rev | GCCTTCATGCTCCTTGATTTCC |
| F-tag amp fwd | GTCGTCATAACGATGAGGTGTTGC |
| F-tag amp rev | ATGAAGCACAGATTCTTCGTTG |
| G-tag amp fwd | GAGAAGAACGGCATAGTGCGTG |
| G-tag amp rev | GTAAGTTTCACGAGGTTCTAC |
| I-tag amp fwd | GCGTCAATCGTATGTGAATGC |
| I-tag amp rev | GCCTTTGAGTGAGCTGATACC |
| ILV2 upstream fwd | TCCTTTCTCCACCATCCCTA |
| ILV2 downstream rev | CGTGTCCGACGAGTTAAAAC |
| Knockout cassette amplification | |
| ILV2 KO fwd | TTTACAAAATCTAAACCCTTTGAGCTAAGAGGAGATAAATACAACAGAATCAATTTTCAACAGCTGAAGCTTCGTACGC |
| ILV2 KO rev | AATAATAATAAAGTCTGCATTTTTTACTGAAAATGCTTTTGAAATAAATGTTTTTGAAATGCATAGGCCACTAGTGGATCTG |
| ILV3 KO fwd | CTGTAATCTTTAGTAACGGATTCTTGTATTTTTTTGTAAACAGCCAAGAAAAAAGTAGAGCAGCTGAAGCTTCGTACGC |
| ILV3 KO rev | AAAGATGATGGAAAAGGAGAATCTCTATATATATATTCATCGATTGGGGCCTATAATGCAGCATAGGCCACTAGTGGATCTG |
| ILV5 KO fwd | AACCTATTCCTAGGAGTTATATTTTTTTACCCTACCAGCAATATAAGTAAAAAATAAAACCAGCTGAAGCTTCGTACGC |
| ILV5 KO rev | ACTTGATGTTGCAAAAATTCCAAGAGAAAAAGTTTCCAGCACTTGATATTATTTTCCTCTGCATAGGCCACTAGTGGATCTG |
| ILV6 KO fwd | TACATAGTTCGTATATACAGAATCTTTAGAACATCTGAGCTCACTAACCCAGTCTTTCTACAGCTGAAGCTTCGTACGC |
| ILV6 KO rev | TACGTTATATAGATGTATAGAGGAGAGTCCCGAGGGCGATCGCAAGGCCGAGAGACTAACGCATAGGCCACTAGTGGATCTG |
| Knockout conformation | |
| ILV2 upstream fwd | TCCTTTCTCCACCATCCCTA |
| ILV2 downstream rev | CGTGTCCGACGAGTTAAAAC |
| ILV3 upstream fwd | CCCTCTTGTATCCATTCC |
| ILV3 downstream rev | CTTTAGTGGCAGCAAAGC |
| ILV5 upstream fwd | GTTGTGCGCGTGCACATTTC |
| ILV5 downstream rev | AATCGTAGCTGTCCCGATGAGG |
| ILV6 upstream fwd | GCACATCCAACGAATCACCTCACCGTTATC |
| ILV6 downstream rev | CGCGTCACCTCGTACAAACGTACAATC |
| Verification of plasmid transformation | |
| GPD1 promoter Fwd | GGGATGTGCTGCAAGGCGATTAAGTTGG |
| CYC1 terminator Rev | GGCAGTGAGCGCAACGCAATTAATGTGAG |
| Cassette integration | |
| ilvD amp with ILV2 hom fwd | TTTACAAAATCTAAACCCTTTGAGCTAAGAGGAGATAAATACAACAGAATCAATTTTCAAGCCAGAGGTATAGACATAGCCAGAC |
| ilvD amp (I-tag rev) | AGACGTCGCGGTGAGTTCAG |
| hphNT1 amp with I-tag hom fwd | TATTCACGTAGACGGATAGGTATAGCCAGACATCAGCAGCATACTTCGGGAACCGTAGGCCCAGCTGAAGCTTCGTACGC |
| hphNT1 amp with B-tag hom rev | GTTGAACATTCTTAGGCTGGTCGAATCATTTAGACACGGGCATCGTCCTCTCGAAAGGTGGCATAGGCCACTAGTGGATCTG |
| ilvB amp (B-tag fwd) | TACTCGCCGATAGTGGAAAC |
| ilvB amp (C-tag rev) | CGCGTGTACGCATGTAAC |
| ilvN amp (C-tag fwd) | GCAAATGCCTGCAAATCG |
| ilvN amp (D-tag rev) | GCCTTCATGCTCCTTGATTTCC |
| ilvC amp (D-tag fwd) | GCTAAATGTACGGGCGACAG |
| ilvC amp with ILV2 hom rev | AATAATAATAAAGTCTGCATTTTTTACTGAAAATGCTTTTGAAATAAATGTTTTTGAAATTGCCGAACTTTCCCTGTATGAAGC |