TABLE 2.
Primers used in this work
| Use | Primer | Nucleotide sequence (5′-3′)a |
|---|---|---|
| Construction of mutant | alpV-replace1 | GTCATGGGTGGGGCCCGTGACGGAGGGTGCGGGGTGTCATGTAGGCTGGACCTGCTTC |
| strains and control | alpV-replace2 | GATCACCCGGCCTGCGGCCTGACTCATGGGGGAAACGTGATTCCGGGGATCCGTCGACC |
| of gene replacement | CV1 | TTGACAAACCGACTGTGC |
| CV2 | TCATGTGGAGCTGCTGC | |
| Complementation | AlpV-amplify1 | CGCGGATCCGGCACGTCATGGGTG |
| AlpV-amplify2 | GCGAATTCAATAAGACCGCAAGGTCTGTTC | |
| Construction of | Kan-1 | ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCATTCCGGGGATCCGTCGACC |
| conjugative cosmids | Kan-2 | TCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGTGTAGGCTGGAGCTGCTTC |
| Real-time and RT- | alpV-1 | GAAGTGCTGGGGACGCTG |
| PCR | alpV-2 | TGGTCGGCGTTGAGGG |
| alpT-1 | GAGCAGAGCCGCCTGGT | |
| alpT-2 | CCAGGTCGGTGAGTTCGG | |
| alpU-1 | CAGATTCACGACGAGCGG | |
| alpU-2 | CCACAACTCGTCCACCAGG | |
| alpZ-1 | AGGAACTGCGTGCGGAGT | |
| alpZ-2 | CAGCCACGCACTCTCGG | |
| hrdB-F | CGCGGCATGCTCTTCCT | |
| hrdB-R | AGGTGGCGTACGTGGAGAAC | |
| KSI-F | CCACGACGAACCCGAGTG | |
| KSI-R | GTAGGCGTTGGAGCGGGT |
a
For the primers alpV-replace1 and alpV-replace2, the sequences in boldface type correspond to the sequences immediately upstream (alpV-replace2) and downstream (alpV-replace1) of alpV, and include the stop and start codons (underlined). For Kan-1 and Kan-2, the sequences in boldface type correspond to the sequences upstream (Kan-1) and downstream (Kan-2) of the neo gene from SuperCos1 (11). The 3′ ends of these four primers match a different end of the resistance cassette. For the primers AlpV-amplify1 and AlpV-amplify2, the sequences in boldface type correspond, respectively, to the BamHI and EcoRI sites used for cloning into pSET152.