TABLE 2.
PCR primersa
| Primer no. | Sequence | Description |
|---|---|---|
| 3554 | 5′-TATGAGCGATAAAATACTTATTGTG | VanR forward primer 1 |
| 3546 | 5′-TGAGCGATAAAATACTTATTGTG | VanR forward primer 2 |
| 3545 | 5′-TTTTTTCAATTTTATAACCAAC | VanR reverse primer 1 |
| 3547 | 5′-AGCTTTTTTTCAATTTTATAACCAAC | VanR reverse primer 2 |
| 3305 | 5′-TATGTTGGTTATAAAATTGAAAAA | VanS forward primer 1 |
| 3307 | 5′-TGTTGGTTATAAAATTGAAAAATA | VanS forward primer 2 |
| 4913 | 5′-TTAGGACCTCCTTTTATCAACCAA | VanS reverse primer 1 |
| 4914 | 5′-GATCTTAGGACCTCCTTTTATCAACCAA | VanS reverse primer 2 |
a
To construct the VanR NdeI-HindIII cloning cassette, two flush amplimers were obtained, with E. faecium A634 DNA used as the template, Pfu DNA polymerase, and primer pairs 3554-3545 and 3546-3547. The two flush amplimers were gel purified, melted, and annealed to produce the desired cloning cassette with NdI- and HindIII-compatible cohesive ends. The VanS cassette was constructed similarly.