TABLE 2.
Oligonucleotide primers used in this study
| Purpose | Directiona | 5′→3′ sequenceb | Equivalent position in pWW0 sequence (accession number AJ344068) |
|---|---|---|---|
| orf1 probe for blotting | Reverse | GGCGCCGGCGGTGATGAC | 85778-85761 |
| Forward | AGGATGCGGCAGCGACGAATAAAG | 84725-84748 | |
| External primer for OEMc of tnpA | Reverse | GATTACGAATTCGAGCTCGGTACCd | Derived from MCS of pGPTKm2 |
| Forward | TGATATTGCAATTGTCCTCGGTGCCT | 844157-86328 | |
| Internal mutagenesis of tnpA | Reverse | GCCCCAGGAtCc-Δ7-GGCGTTGAAGATGTCGCG | 86844-86808 |
| Forward | TCAACGCC-Δ7-gGaTCCTGGGGCGCTGGCACCG | 86818-86854 | |
| External primer for OEM of IspA and orf1 | Reverse | CCGAGGACAATTGCAATATCAGCCAGG | 86323-86297 |
| Forward | CGGCCTCGAGCAAGACGTTTCCCGT | Derived from Kmr cassette | |
| Internal mutagenesis of lspA | Reverse | CGATATGGAtcc-Δ7-TACGGCGAGAGCTTTTTGCCA | 85938-85898 |
| Forward | CTCTCGCCGTA-Δ7-ggaTCCATATCGGGCCTGCTG | 85947-85908 | |
| Internal mutagenesis of orf1 | Reverse | GCATAAACA-Δ7-ggaTCCCTGGCGCCTCGGAGGAGG | 85093-85054 |
| Forward | GCGCCAGGGAtcc-Δ7-TGTTTATGCCGTGCCGAAG | 85065-85103 | |
| External primer for OEM of orf2 | Reverse | GCGGGCCTCGAGCAAAGACGTTTCCCGTTGA | Derived from Kmr cassette |
| Forward | GCATGCCTGCAGGTCGACCAGTCCCT | Derived from MCS of pGPTKm1 | |
| Internal mutagenesis of orf2 | Reverse | TCGGCA-Δ7-GGaTcCCGTGTCTATACCGAGAAACAC | 84802-84763 |
| Forward | GGTATAGACACGGgAtCC-Δ7-TGCCGATCAGGCGGCG | 84772-84812 | |
| Sequencing out from IRR | Reverse | ACGATGACCGGGAAGTTGAGG | 84554-84534 |
| Sequencing out from IRL | Forward | GCCGGACTTCGCAGAGGA | 87659-87676 |
a
Forward primers operate from left to right relative to Fig. 1.
b
For the external primers for overlap extension mutagenesis, the relevant restriction sites (see Fig. 3) are in boldface. For the internal mutagenesis primers, the deletion of 7 bp is indicated by -Δ7-and the constructed BamHI site is underlined, with the bases which are changed from the wild-type sequence to form it indicated in lowercase letters. Forward primers function from left to right relative to Fig. 1.
c
OEM, overlap extension mutagenesis.
d
The XbaI site used for the overlap extension mutagenesis of tnpA (see Fig. 3) is within the PCR fragment and not on the primer.