TABLE 3.
Oligonucleotides used for PCRs
| Primer and function | Sequence (5′-3′)a |
|---|---|
| Antibiotic resistance | |
| cat-5′ | GGCGGCCGCATGCCTGCAGGTCCAACTTTTGGCG |
| cat-3′ | GGCGGCCGCTTACGCCCCGCCCTGCCACTCATCGC |
| aph5′USER | GGGAAAGUGCGGCCGCTTCTGATTAGAAAAACTCATCGAGC |
| aph3′USER | GGAGACAUGCGGCCGCAAAGCCACGTTGTGTCTCAAAATC |
| tetAR-5′ | GGCGGCCGCGCGCGTTCAATCGGACCAG |
| tetAR-3′ | GGCGGCCGCTAGTGAGTGGGTTGCGCTCC |
| USER friendly constructions | |
| pCVD442-USER Linker top | CTGAGGGAAAGTCTAGATGTCTCCTCAGCATGAGCT |
| pCVD442-USER Linker bottom | CATGCTGAGGAGACATCTAGACTTTCCCTCAGCATG |
| pRK437-USER Linker top | AGCTTGGCTGAGGGAAAGTCTAGATGTCTCCTCAGCGG |
| pRK437-USER Linker bottom | GATCCCGCTGAGGAGACATCTAGACTTTCCCTCAGCCA |
| Flagellin deletion | |
| flaFBAup5′ | GGTCGACAAAAACGCGCGGAAGTGGCG |
| flaFBAup3′ | GGCGGCCGCGGCTTTGGATACTTATCGACCA |
| flaFBAdown5′ | GGCGGCCGCCGGTAGACAGTGAGCGTGCC |
| flaFBAdown3′ | GGAGCTCCTGACGAGCTTGCGCCATT |
| flaCDEup5′ | GGTCGACGTGAACATCGAAGATGTGCC |
| flaCDEup3′ | GGCGGCCGCGCTCACAAAAACAAAAAACGCTG |
| flaCDEdown5′ | GGCGGCCGCTCTCCTTTCGAGTTCGCAAGC |
| flaCDEdown3′ | GGAGCTCGTTGCAAGCTTCGCAGCTCG |
| flaFBAup-out | TGATGGCACTGAAAATCCGAAGC |
| flaFBAdown-out | CATTCATCTTTTCCACCATTCTCAG |
| flaCDEup-out | ATTATCGCACCTTTGTCTGGTGAG |
| flaCDEdown-out | TTGAATTCAATGAGAATTCACGCGC |
| Motility and chemotaxis deletion | |
| motABdown5′ | TGTTTAGCGGCCGCGGATGGGCACTCCTCATGCTATTTTCCG |
| motABdown3′ | GGAGACAUCAGTTGATCCACCAAATTGTCC |
| motABup5′ | GGGAAAGUTGGGTTGTGCAGTTTGACCG |
| motABup3′ | CCCATCCGCGGCCGCTAAACAGGGTACGTCAGTTGAATAAAAG |
| cheYdown5′ | TGTTTAGCGGCCGCGGATGGGAGATTACTTCGTCAGAGCTGC |
| cheYdown3′ | GGAGACAUCAAAGGGCCTTGAATTCACTC |
| cheYup3′ | CCCATCCGCGGCCGCTAAACATGTTTTAATCATTTTGTGTCCAAG |
| cheYup5′ | GGGAAAGUGACGCCTCAAAAGGCGCAAG |
| motABup-out | ATCCACCACTTCAATCTTGATGG |
| motABdown-out | CGTCATCTAAAGCCAGATCACCG |
| cheYup-out | TCCAAGCGGGTATGATTGGCC |
| cheYdown-out | TTGAACTAAAAGATCTTCGATACG |
| RTX deletion | |
| rtxA1-down-3′ | GGGAAAGUCGCTTATGGCAACGGAATTCG |
| rtxA1-down-5′ | ACCCGGGUCAACGAGGCACGAGTATAGAG |
| rtxA1-up-3′ | ACCCGGGUTAAGCCAAACTCTTCTTTAGGAG |
| rtxA1-up-5′ | GGAGACAUGAGCTTGCAGGCGGAGAGTGA |
| rtxC-up5′ | GGAGACAUTTGAGCTTGCAGGCGGAGAG |
| rtxC-up3′ | CTCTTCTTTAGGGCGGCCGCTCTGCCCTATATCAACCAATATG |
| rtxC-down5′ | GATATAGGGCAGAGCGGCCGCCCTAAAGAAGAGTTTGGCTTATG |
| rtxC-down 3′ | GGGAAAGUAGTACGGTCTAGTTTATTGCCC |
| RtxA1-deletion up | GAGCTTGCAGGCGGAGAGTGA |
| RtxA1-deletion down | CGCTTATGGCAACGGAATTCG |
a
Sequences in boldface type indicate engineered restriction enzyme sites, and underlined sequences indicate USER friendly ends for cloning.