TABLE 3.
Oligonucleotide primers used in this study
| Primer namea | Primer sequence 5′–3′ |
|---|---|
| pEGFP-C2 F | AACACCCCCATCGGCG |
| pEGFP-C2 R | GTAACCATTATAAGCTGC |
| NotI | TGCGGCCGCA |
| pTrc99A F | CGGTTCTGGCAAATATTC |
| pTrc99A R | GCAGTTCCCTACTCTCGC |
| P63A Fb | GCGATACGAAAAAGGAGAAGTAGCAATATTGAATACCAAAGAACATCCG |
| P63A Rb | CGGATGTTCTTTGGTATTCAATATTGCTACTTCTCCTTTTTCGTATCGC |
| I64A Fb | GCGATACGAAAAAGGAGAAGTACCAGCATTGAATACCAAAGAACATCCG |
| I64A Rb | CGGATGTTCTTTGGTATTCAATATTGCTACTTCTCCTTTTTCGTATCGC |
| L65A Fb | GCGATACGAAAAAGGAGAAGTACCAATAGCGAATACCAAAGAACATCCGTATTTG |
| L65A Rb | CAAATACGGATGTTCTTTGGTATTCGCTATTGGTACTTCTCCTTTTTCGTATCGC |
| N66A Fb | GATACGAAAAAGGAGAAGTACCAATATTGGCTACCAAAGAACATCCGTATTTGAGC |
| N66A Rb | GCTCAAATACGGATGTTCTTTGGTAGCCAATATTGGTACTTCTCCTTTTTCGTATC |
| K68A Fb | GAAGTACCAATATTGAATACCGCAGAACATCCGTATTTGAGC |
| K68A Rb | GCTCAAATACGGATGTTCTGCGGTATTCAATATTGGTACTTC |
| K81A Fb | GAGCAATATTATAAATGCTGCAGCAATAGAAAATGAGCGTATAATCGG |
| K81A Rb | CCGATTATACGCTCATTTTCTATTGCTGCAGCATTTATAATATTGCTC |
| D93A Fb | GTATAATCGGTGTGCTGGTAGCTGGAAATTTTACTTATGAACAAAAAAAG |
| D93A Rb | CTTTTTTTGTTCATAAGTAAAATTTCCAGCTACCAGCACACCGATTATAC |
| D121A Fb | CAAAATATAAAAATAATCTACCGAGCAGCTGTGGATTTCAGCATGTATG |
| D121A Rb | CATACATGCTGAAATCCACAGCTGCTCGGTAGATTATTTTTATATTTTG |
| D128A Fb | CAGATGTGGATTTCAGCATGTATGCTAAAAAACTATCTGATATTTAC |
| D128A Rb | GTAAATATCAGATAGTTTTTTAGCATACATGCTGAAATCCACATCTG |
| L157A Fb | GAGGGATAATTATCTGTTAGGCGCATTAAGAGAAGAGTTAAAAAATATCC |
| L157A Rb | GGATATTTTTTAACTCTTCTCTTAATGCGCCTAACAGATAATTATCCCTC |
| Y219A Fb | CGCCCTGTAGCGGATGTATAGCTCTTGATGCCGACATGATTATTAC |
| Y219A Rb | GTAATAATCATGTCGGCATCAAGAGCTATACATCCGCTACAGGGCG |
| Y234A Fb | CCGATAAATTAGGAGTCCTGGCTGCTCCTGATGGTATCGCTGTG |
| Y234A Rb | CACAGCGATACCATCAGGAGCAGCCAGGACTCCTAATTTATCGG |
| P236A Fb | GGAGTCCTGTATGCTGCTGATGGTATCGCTGTGC |
| P236A Rb | GCACAGCGATACCATCAGCAGCATACAGGACTCC |
| D237A Fb | GGAGTCCTGTATGCTCCTGCTGGTATCGCTGTGCATGTAG |
| D237A Rb | CTACATGCACAGCGATACCAGCAGGAGCATACAGGACTCC |
| G238A Fb | GGAGTCCTGTATGCTCCTGATGCTATCGCTGTGCATGTAG |
| G238A Rb | CTACATGCACAGCGATAGCATCAGGAGCATACAGGACTCC |
| I239A Fb | GGAGTCCTGTATGCTCCTGATGGTGCCGCTGTGCATGTAG |
| I239A Rb | CTACATGCACAGCGGCACCATCAGGAGCATACAGGACTCC |
| H242A Fb | CTCCTGATGGTATCGCTGTGGCTGTAGATTGTAATGATGAG |
| H242A Rb | CTCATCATTACAATCTACAGCCACAGCGATACCATCAGGAG |
| E253A Fb | GTGGATTGTAATGATAATAGAAAAAGTCTTGCAAATGGTGCAATAGTTGTCAATC |
| E253A Rb | GATTGACAACTATTGCACCATTTGCAAGACTTTTTCTATTATCATTACAATCCAC |
| N263A Fb | GTCAATCGTAGTGCTCATCCAGCATTACTTGCAGGCCTCGATATTATG |
| N263A Rb | CATAATATCGAGGCCTGCAAGTAATGCTGGATGAGCACTACGATTGAC |
| L267A Fb | GTAATCATCCAGCAGCACTTGCAGGCCTCGATATTATGAAGAG |
| L267A Rb | CTCTTCATAATATCGAGGCCTGCAAGTGCTGCTGGATGATTAC |
| A269E Fb | GTAATCATCCAGCATTACTTGAAGGCCTCGATATTATGAAGAG |
| A269E Rb | CTCTTCATAATATCGAGGCCTTCAAGTAATGCTGGATGATTAC |
| Y283A Fb | GTAAAGTTGACGCTCATCCAGCTTATGATGGTCTAGGAAAGGG |
| Y283A Rb | CCCTTTCCTAGACCATCATAAGCTGGATGAGCGTCAACTTTAC |
| K292A Fb | GATGGTCTAGGAAAGGGTATCGCGCGGCATTTTAACTATTCATCG |
| K292A Rb | CGATGAATAGTTAAAATGCCGCGCGATACCCTTTCCTAGACCATC |
| PILN Fb | GCGATACGAAAAAGGAGAAGTAGCAGCAGCGGCTACCAAAGAACATCCGTATTTG |
| PILN Rb | CAAATACGGATGTTCTTTGGTAGCCGCTGCTGCTACTTCTCCTTTTTCGTATCGC |
| HKQ Fb | CTGATATTTACCTTGAAAATATCGCTGCAGCAGAATCATACCCTGCCAGTGAGAGGG |
| HKQ Rb | CCCTCTCACTGGCAGGGTATGATTCTGCTGCAGCGATATTTTCAAGGTAAATATCAG |
| QES Fb | CCTTGAAAATATCCATAAAGCAGCAGCATACCCTGCCAGTGAGAGGG |
| QES Rb | CCCTCTCACTGGCAGGGTATGCTGCTGCTTTATGGATATTTTCAAGG |
| LGLL Fb | CAGTGAGAGGGATAATTATCTGGCAGCCGCAGCAAGAGAAGAGTTAAAAAATATCCCAGAAGG |
| LGLL Rb | CCTTCTGGGATATTTTTTAACTCTTCTCTTGCTGCGGCTGCCAGATAATTATCCCTCTCACTG |
| GSL Fb | GGCCATATTGAAGGCTGCAGCTGCGTTTACAGAGACGGGAAAAACTGG |
| GSL Rb | CCAGTTTTTCCCGTCTCTGTAAACGCAGCTGCAGCCTTCAATATGGCC |
| DKL Fb | GCCGACATGATTATTACCGCTGCAGCAGGAGTCCTGTATGCTCCTG |
| DKL Rb | CAGGAGCATACAGGACTCCTGCTGCAGCGGTAATAATCATGTCGGC |
| PDG Fb | GATAAATTAGGAGTCCTGTATGCTGCTGCTGCTATCGCTGTGCATGTAGATTG |
| PDG Rb | CAATCTACATGCACAGCGATAGCAGCAGCAGCATACAGGACTCCTAATTTATC |
| SN Fb | GGTGCGATAGTTGTCAATCGTGCTGCTCATCCAGCATTACTTGC |
| SN Rb | GCAAGTAATGCTGGATGAGCAGCACGATTGACAACTATCGCACC |
| LAGL Fb | GTCAATCGTAGTAATCATCCAGCATTAGCTGAAGCCGCCGATATTATGAAGAGTAAAGTTGACGC |
| LAGL Rb | GCGTCAACTTTACTCTTCATAATATCGGCGGCTTCAGCTAATGCTGGATGATTACTACGATTGAC |
| KV Fb | GGCCTCGATATTATGAAGAGTGCAGCTGACGCTCATCCATATTATG |
| KV Rb | CATAATATGGATGAGCGTCAGCTGCACTCTTCATAATATCGAGGCC |
| KGI Fb | CATCCATATTATGATGGTCTAGGAGCGGCTGCCAAGCGGCATTTTAACTATTCATCG |
| KGI Rb | CGATGAATAGTTAAAATGCCGCTTGGCAGCCGCTCCTAGACCATCATAATATGGATG |
| B1 F | CCGGTACCATGTTATCTTCATTAAATGTCC |
| B1 R | C GGAATTCCC CCATGAACTGCTGGTATAC |
| pCX340 F | AAGGCGCACTCCCGTTC |
| pCX340 R | GTCATTCTGAGAATAGTG |
| pGEX F | CGTATTGAAGCTATCCCACAA |
| pGEX R | GGGAGCTGCATGTGTCAGAG |
| pGEX B1 F | CGCGAATTCATGTTATCTTCATTAAATGTCCTTC |
| pGEX B1 R | CGCGTCGACTTACCATGAACTGCTGGTATAC |
| CR B F | CGGGATCCGGATAAACAGGACGACGAATGTTATCTCCATTAAATGTTCTTCAATTTAATTTC |
| CR B R | CGGTCGACTTACCATGAACTGTTGGTATACATACTGG |
| pACYC seq F | GTACTGCCGGGCCTCTTG |
| pACYC seq R | GAAGGCTTGAGCGAGGGCG |
| CR Y219A F | CTCCATGTGGGGGATGTATAGCTCTTGATGCAGATATGATTATTAC |
| CR Y219A R | GTAATAATCATATCTGCATCAAGAGCTATACATCCCCCACATGGAG |
| CR E253A F | GTGGATTGTAATGATAATAGAAAAAGTCTTGCAAATGGTGCAATAGTTGTCAATC |
| CR E253A R | GATTGACAACTATTGCACCATTTGCAAGACTTTTTCTATTATCATTACAATCCAC |
a
F, forward primer; R, reverse primer.
b
Primers used to introduce site-directed mutations in nleB1. The primer name is indicative of the amino acid mutation(s) introduced in NleB1.