TABLE 1.
Oligonucleotide primers and plasmids used in this study
| Primer or plasmid | Sequence or description | Reference |
|---|---|---|
| Primera | ||
| SELGTAR 1U | 5′-GGGCCCGTCATCTCCTTGAGATTCGTG-3′ | |
| SELGTAR 2L | 5′-GATTCTTGCTCCAATAATTGCC-3′ | |
| SELGTAR 3U | 5′-GGCAATTATTGGAGCAAGAATCGTTTCTCAATACATGTCGGTG-3′ | |
| SELGTAR 4L | 5′-CCGCGGATAATTTAGAAGCGACCTTGC-3′ | |
| 5′PRTM | 5′-GGGGAATTCAAGTGTCATTACGATGAAGG-3′ | |
| PRTM-NDEL | 5′-GGGGATATCGTAATCAGCATCTGTCAGCTC-3′ | |
| 3′PRTM | 5′-GGGGTCGACTTTCTGACTTAGATTTAGAAG-3′ | |
| PRTM-CDEL | 5′-GGGGATATCGAGGGTGATATTTCAGAGGTG-3′ | |
| Plasmid | ||
| pG+host9:ISS1 | Replication-thermosensitive derivative of pWV01 containing cloned ISS1 sequence | 29a |
| pAH08 | pG+host9:ISS1b containing in-frame-deleted lgt gene of S. equi | This study |
| pGprtMΔ | pG+host9:ISS1b ISS1 containing in-frame-deleted prtM gene of S. equi | This study |
| pGEM-T | T-A cloning vector | Promega |
a
Underlined sequences represent engineered restriction sites for ApaI (GGGCCC), SacII (CCGCGG), EcoRI (GAATTC), EcoRV (GATATC), and SalI (GTCGAC). The italicized sequence in SELGTAR 3U is the reverse and complementary sequence to the primer SELGTAR 2L sequence and provides the overlapping sequence for the overlap-deletion PCR strategy.
b
The ISS1 sequence is completely removed during cloning strategy.