Table 1

Oligonucleotides used to construct plasmids for expression of hph genes and pheA and aroG genes

PlasmidCommentPrimerSequencea
pHPH01 (pTrc99a-Ptrc-hphCD)hphCD_Fw_EcoRI5′-ATAGAATTCAGTCACGAAAATAACGTTTTG-3′
hphCD_Rv_KpnI5′-AGAGGTACCTTATTATTAATCAAAGCGATCGCTAATTTTTC-3′
pHPH02 (pTrc99a-Ptrc-hphCD-hphA)Derived from pHPH01hphA_Fw_KpnI5′-AGAGGTACCAAGAGGAGAAATTAACTATGGAAACCCTTCCTATC-3′
hphA_Rv_BamHI5′-AGAGGATCCTGATGAATTATTTAAACACCCCATTTTTC-3′
pHPH03 (pSTV29-Plac-hphB)hphB_Fw_BamHI5′-AGAGGATCCAAGAGGAGAAATTAACTATGGAATCTTTAAGTAAGG-3′
hphB_Rv_PstI5′-AGACTGCAGTTATTATTATTTTCGTGACTCATGAAGTAC-3′
pHPH04 (pSTV29-Ptrc-hphB)Ptrc-hphB fragment was amplified from pHPH08Ptrc_Fw_SacI5′-ACAGAGCTCGCATAATTCGTGTCGCTCAAG-3′
hphB_Rv_PstI5′-AGACTGCAGTTATTATTATTTTCGTGACTCATGAAGTAC-3′
pHPH05 (pTrc99a-Ptrc-hphA)hphA_Fw_NcoI5′-ACTACACCATGGAAACCCTTCCTATC-3′
hphA_Rv_BamHI5′-AGAGGATCCTGATGAATTATTTAAACACCCCATTTTTC-3′
pHPH06 (pTrc99a-Ptrc-hphA-hphB)Derived from pHPH05hphB_Fw_BamHI5′-AGAGGATCCAAGAGGAGAAATTAACTATGGAATCTTTAAGTAAGG-3′
hphB_Rv_PstI5′-AGACTGCAGTTATTATTATTTTCGTGACTCATGAAGTAC-3′
pHPH07 (pSTV29-Ptrc-hphCD)Ptrc-hphCD fragment was amplified from pHPH01Ptrc_Fw_SacI5′-ACAGAGCTCGCATAATTCGTGTCGCTCAAG-3′
hphCD_Rv_KpnI5′-AGAGGTACCTTATTATTAATCAAAGCGATCGCTAATTTTTC-3′
pHPH08 (pTrc99a-Ptrc-hphB)hphB_Fw_NcoI5′-ACTACACCATGGAATCTTTAAGTAAGG-3′
hphB_Rv_EcoRI5′-ATAGAATTCTTATTATTATTTTCGTGACTCATGAAGTAC-3′
pHPH09 (pTrc99a-Ptrc-hphB-hphA)Derived from pHPH08hphA_Fw_KpnI5′-AGAGGTACCAAGAGGAGAAATTAACTATGGAAACCCTTCCTATC-3′
hphA_Rv_BamHI5′-AGAGGATCCTGATGAATTATTTAAACACCCCATTTTTC-3′
pHPH10 (pTrc99a-Ptrc-hphA-hphCD)Derived from pHPH05hphCD_Fw_BamHI5′-AGAGGATCCAAGAGGAGAAATTAACTATGAGTCACGAAAATAACG-3′
hphCD_Rv_HindIII5′-AAAAGCTTTTATTATTAATCAAAGCGATCGCTAATTTTTC-3′
pHPH11 (pTrc99a-Ptrc-hphB-hphCD)Derived from pHPH08hphCD_Fw_EcoRI5′-ATAGAATTCAGTCACGAAAATAACGTTTTG-3′
hphCD_Rv_KpnI5′-AGAGGTACCTTATTATTAATCAAAGCGATCGCTAATTTTTC-3′
pHPH12 (pSTV29-Ptrc-hphA)Ptrc-hphA fragment was amplified from pHPH05Ptrc_Fw_SacI5′-ACAGAGCTCGCATAATTCGTGTCGCTCAAG-3′
hphA_Rv_BamHI5′-AGAGGATCCTGATGAATTATTTAAACACCCCATTTTTC-3′
pMWF01 [pMW219-Ptrp-pheAfbr-aroG(P150L)]pTrp_Fw_EcoRI5′-ATCGGAATTCTCATGTTTGACAGCTTATCATCG-3′
pTrp_Rv_BamHI5′-ATCGGGATCCGTGAACTTGCGTACTAGTTAAC-3′
pheA_Fw_BamHI5′-ATCGGGATCCAAAAAGGCAACACTATGACATCGGA-3′
pheA330_Rv_SalI5′-ATCGGTCGACTTATGATTCCAGACGGGTCATAATC-3′
aroG_Fw_SalI5′-ATCGGTCGACAAAGGAACAGACATGAATTATCAGA-3′
aroG_Rv_SphI5′-ATCGGCATGCATCCGACAATTAAACCTTACCCG-3′
aroG_P150L_15′-CTTTCTCGATATGATCACCCtgCAATATCTC-3′
aroG_P150L_25′-CAGGTCAGCGAGATATTGcaGGGTGATCAT-3′
  • a Italics indicate sites digested by restriction enzymes; lowercase indicates the mutational site.