TABLE 2.
Oligonucleotidesa (5ʹ‐3ʹ) used in this study
| Oligonucleotides (5′‐3′) used in this study | |
|---|---|
| BACTH | |
| Pst_pKT25_ToxR_fwd_BACTH | ATTCTGCAGTCGGATTAGGACACAACTC |
| Pst_pUT18C_ToxR_fwd_BACTH | ATTCTGCAGTTTCGGATTAGGACACAACT |
| XbaI_ToxR_rev_BACTH | ATTTCTAGACTACTCACACACTTTGATGG |
| ToxS_XbaI‐fwd | TAATCTAGAGATGCAAAATAGACACATCGCC |
| ToxS‐FLAG_EcoRI‐rev | TATGAATTCTGAAAATCTTCTCTCACTCGA |
| XbaI_ToxS_rev | ATTTCTAGATTAAGAATTACTGAACAGTACG |
| BamHI_ompUO123_fwd | ATTGGATCCTCCTAAATCGGGTCGGGT |
| KpnI_ompUO123_rev | AAAGGTACCATTGGTCATTGTTGTGTTCA |
| toxRC236S and toxRC293S substitution | |
| HindIII_toxR_5ʹ_FLAG | AATAAGCTTATGTTCGGATTAGGACACAACTCA |
| KpnI_toxR_3ʹ_FLAG | AATGGTACCCTACTCACACACTTTGATGGCAT |
| KpnI_toxR293S_3ʹ_FLAG | AATGGTACCCTACTCAGACACTTTGATGGCATCGTTA |
| toxRC236S_5ʹ | GGCTACCGTCAATCGAACTGAGCGTTAAAAAATACAATGA |
| toxRC236S_3ʹ | TCATTGTATTTTTTAACGCTCAGTTCGATTGACGGTAGCC |
| SacI_toxRS_1 | TTTGAGCTCATTTGGAAATCACATCGCGCAAAC |
| XbaI_toxRS_4 | TTTTCTAGAATGACGTTTCCCCGCGGTGAG |
| c_FLAGtoxR_3ʹ_F1 | TGTCATCGTCGTCCTTGTAGTCCATCTAATGTCCCAGTATCTCCCTGT |
| c_FLAGtoxR_5ʹ_F2 | GGGACAGGGAGATACTGGGACATTAGATGGACTACAAGGACGACGATGA |
| c_FLAGtoxR_3ʹ_F2 | CTACTCACACACTTTGATGGCAT |
| c_FLAGtoxRC293S_3ʹ_F2 | CTACTCAGACACTTTGATGGCAT |
| c_FLAGtoxR_5ʹ_F3 | AACCAGTTAACGCTGAATTACATTC |
| c_FLAGtoxRC293S_5ʹ_F3 | GTTGCTAACCCTAACGATGCCATCAAAGTGTCTGAG |
| toxRC293SW76R substitution | |
| F1_ToxRC293S‐W76R_XbaI_fwd | TTATCTAGAATCCGCCACGATGAAAGCCGA |
| F1_SOE_ToxRC293S‐W76R_rev | TTGCTCTCGCCGAACAAAGTCATGCAAATCATTGCGAGA |
| F2_SOE_ToxRC293S‐W76R_fwd | GACTTTGTTCGGCGAGAGCAAGGTTTTGAAGTCGATGAT |
| F2_ToxRC293S‐W76R_SacI_rev | TAAGAGCTCCAGACCGCAGCATCCAATTGC |
| pBAD18‐KanFLAGtoxRS, pBAD18‐FLAGtoxRS, pFLAGtoxRW76RS | |
| fwd_SacI_pFlagMAC_ShineD | TTAGAGCTCATAACAATTTCACACAGGAGA |
| FLAGtoxR_fw_KpnI | ATAGGTACCATGTTCGGATTAGGACACAACTCA |
| BglII_toxRS_3ʹFLAG | TTAAGATCTTTAAGAATTACTGAACAGTACGGT |
| Sequencing | |
| phoA‐seq‐rev | GCTCACCAACTGATAACCAC |
| SacIDsbA1 | TTTGAGCTCCAAGAAGAGATCCCGATCGTC |
| Kan_cassette_rv | TTAGAAAAACTCATCGAGCA |
| PhoA3ʹ 180 rev | GCTAAGAGAATCACGCAGA |
| pGP704_CVD_rv|15 | GATGTAACGCACTGAGAAG |
| pBAD_fwd | CCATAGCATTTTTATCCATAAG |
Restriction sites are underlined. Bold letters indicate codons changed to obtain desired amino acid mutations.