Table 1.
Primer sequencesa
| Primer function and name | Primer sequence (5′ to 3′) | Size of amplicon (bp) |
|---|---|---|
| Replacement of FgMET12 and FgMET13 | ||
| Fg-MET12-A1 | CCAGTGAATTCGAGCTCGGTACCAAGGCCCAGAGCTCCCTTCCAGTCCATCC | 1,198 |
| Fg-MET12-A2 | CTTGCGCGCCTAGGCGGCCGTGGCCAGCCCGAAGTTCTGTCAAGTTAAGCAAGGTGTCTA | |
| Fg-MET12-A3 | GGCCGGCCGGCGCGCCGTTTAAACGGATTTATGGGGTATTTATTCGTGGTATGTGAGTAG | 1,563 |
| Fg-MET12-A4 | GCATGCCTGCAGGTCGACATTAATTAATTTGCCAGTTGATTTTCCCAGCCAGTTCTTT | |
| Fg-MET13-A1 | CCAGTGAATTCGAGCTCGGTACCAAGGCCCAACCGCGCCATTTCCATTTTATCATTACC | 1,614 |
| Fg-MET13-A2 | CTTGCGCGCCTAGGCGGCCGTGGCCAGCCCGGGGGAGGGGAACTGCAACGGAAACTAT | |
| Fg-MET13-A3 | GGCCGGCCGGCGCGCCGTTTAAACGGATTTCGGCGGGCGCAGACGACTTTATGT | 1,746 |
| Fg-MET13-A4 | GCATGCCTGCAGGTCGACATTAATTAATTTCGGTGCGCGAGCTCCTTTTTCTGC | |
| Verification and RT-PCR | ||
| Fg-MET12-T1 (CDS)c | CCTCTAGGCCTGTCGCTTCCCACTCG | 593 (in wild type) |
| Fg-MET12-T2 (CDS) | CGGCGCTGACTTCATACAAACCCAACTC | No product in FgΔmet12 |
| Fg-MET12-T3 | CAGGTCGAGTGGTGAATACTTCG | 1,857 (with T3/RF-1) |
| Fg-MET12-T4 | GCCCTCAGAAATCGCTGCCT | 1,804 (with T4/RF-2) |
| Fg-MET13-T1 (CDS) | CCGACGACAACAAGAACGAGGACGAAC | 598 (in wild type) |
| Fg-MET13-T2 (CDS) | CCAAAAGCGGGAGAGCGAGAATCACC | No product in FgΔmet13 |
| Fg-MET13-T3 | CATCCAATCCCATGAATCCTTG | 1999 (with T3/RF-2) |
| Fg-MET13-T4 | GTTATACGGATCGCGGATCGC | 1994 (with T4/RF-1) |
| Hyg588U | AGCTGCGCCGATGGTTTCTACAA | 588 (with Hyg588U/Hyg588L) |
| Hyg588L | GCGCGTCTGCTGCTCCATACAA | |
| RF-1 | AAATTTTGTGCTCACCGCCTGGAC | NAb |
| RF-2 | TCTCCTTGCATGCACCATTCCTTG | NA |
| Probe for Southern analysis | ||
| Hyg789U | AATAGCTGCGCCGATGGTTTCT | 789 |
| Hyg789L | GCTTCTGCGGGCGATTTGTGTA | |
| Complementation of Scmet13D strain | ||
| Fg-MET12-H1 | AGTGGATCCAACATGGACAAGATCACCGACCGCATCG | 2,019 |
| Fg-MET12-H2 | AGTGCGGCCGCTTAACTTGACAGAACTTCCCATAGA | |
| Fg-MET13-H1 | AGTGGATCCAACATGCATATCAAGGATATGCTCAACG | 1,866 |
| Fg-MET13-H2 | AGTGCGGCCGCTTAGTTCGCAGCGGCAGTGACGGCA |
a
Primers for construction of replacement vectors include 30-bp overhangs, shown in boldface, for Xi cloning.
b
NA, not applicable (used for sequencing).
c
CDS, coding sequence.