TABLE 2.
Oligonucleotides used for this study
| Primer | Sequence (5′→3′)a | Purpose |
|---|---|---|
| RT-rpoD-F | GATCCGGAACAGGTGGAAGAC | RT-PCR of rpoD |
| RT-rpoD-R | TCAGCAGTTCCACGGTACCC | RT-PCR of rpoD |
| RT-mexX-F | CATCAGCGAACGCGAGTACAC | RT-PCR of mexX |
| RT-mexX-R | CAATTCGCGATGCGGATTG | RT-PCR of mexX |
| RT-PA5471-F | ACAGCACCTGGATCGAAGGC | RT-PCR of PA5471 |
| RT-PA5471-R | TTCGATGCAGTCGCTCCAG | RT-PCR of PA5471 |
| RT-PA5470-F | GATCCTGCTGCAACTCTCCG | RT-PCR of PA5470 |
| RT-PA5470-R | ACCAGTTCTTGCGCGCAT | RT-PCR of PA5470 |
| RT-mexZ-F | AAACCCGCGACGGCATACT | RT-PCR of mexZ |
| RT-mexZ-R | ACTGGCGGAGAAAGCCCAT | RT-PCR of mexZ |
| E-PA5471-F | GCTAGAATTCGATCTACCGTTTCAATCACATGGAT; EcoRI | PCR cloning of PA5471 |
| X-PA5471-R | GATCTCTAGAGGCCACCTCCTCGATTACCT; XbaI | PCR cloning of PA5471 |
| EH-mexX-F | GAATTCAAGCTTCAAGCTCGCGAGTTCACGA; EcoRI, HindIII | PCR cloning of mexXY |
| N-mexX-R | ACGTTGGACGAGGCGATCTC | PCR cloning of mexXY |
| mexZ-F | TCGTGAACTCGCGAGCTTG | PCR cloning of mexZ |
| mexZ-R | CACATCAGCGAGGAAGACGC | PCR cloning of mexZ |
| PA5471DU-F | GATCAAGCTTCCTGGGAAGGCTATACCAACGb | Deletion of PA5471; upstream fragment |
| PA5471DU-R | GCTAGGTACCGCCCATAATCCAATCCATGTGb; KpnI | Deletion of PA5471; upstream fragment |
| PA5471DD-F | GCTAGGTACCCGGAAGCCGGTGCTGACCTACA; KpnI | Deletion of PA5471; downstream fragment |
| PA5471DD-R | GCTAGAATTCGCTTCATCGGCACCATCAT; EcoRI | Deletion of PA5471; downstream fragment |
| XPA5470-F | GATCTCTAGATGCTCGACATCAACCACAACC | Deletion of PA5471-PA5470 |
| PA5470-DDR | GCTAGAATTCCCAACGACGCCTTCTACTAC; EcoRI | Deletion of PA5471-PA5470 |
| mexZDU-F | GATCAAGCTTAATTCGCGATGCGGATTG; HindIII | Deletion of mexZ; upstream fragment |
| mexZDU-R | GATCTCTAGACACTGAACGTCCTCACAAGGG; XbaI | Deletion of mexZ; upstream fragment |
| mexZDD-F | GATCTCTAGACGCAGTTCTCCCTCCTGTTG; XbaI | Deletion of mexZ; downstream fragment |
| mexZDD-R | GCTAGAATTCGAAGGAAATCTTGGTGGCGA; EcoRI | Deletion of mexZ; downstream fragment |
| PA0826.2DU-F | GATCAAGCTTTCGAATACCGCCTGCAAGC; HindIII | Deletion of ssrA; upstream fragment |
| PA0826.2DU-R | CTAGTCTAGACCGGCGTCGAATCCTAATC; XbaI | Deletion of ssrA; upstream fragment |
| PA0826.2DD-F | CTAGTCTAGAGCATGTAGAACCGATAGCGGA; XbaI | Deletion of ssrA; downstream fragment |
| PA0826.2DD-R | GCTAGGTACCCAGTCCTTCCTGGCGGCTAT; KpnI | Deletion of ssrA; downstream fragment |
| PA4768DU-F | GCTAGAATTCGCGGTAGATCTCCACCCGTT; EcoRI | Deletion of smpB; upstream fragment |
| PA4768DU-R | GATCTCTAGAGACCATAGGCGGCGCATTATAG; XbaI | Deletion of smpB; upstream fragment |
| PA4768DD-F | GATCTCTAGAGACTTCGACAAGCGCCACAC; XbaI | Deletion of smpB; downstream fragment |
| PA4768DD-R | GATCAAGCTTAGAGGCTTTGCGACGAAACTT; HindIII | Deletion of smpB; downstream fragment |
a
In some instances, restriction sites were introduced into oligonucleotides to be used for PCR, and these are underlined in the sequences, with the corresponding restriction endonucleases indicated.
b
The PCR product amplified with these primers and cloned into pCR-BluntII-TOPO was excised following PstI-KpnI digestion (a PstI site is present within the pCR-BluntII-TOPO multicloning site) prior to cloning into pEX18Tc.