Table 2 Oligonucleotides used in this study
Oligonucleotide | Sequence | Purpose |
|---|---|---|
hisG1 | 5′-CGCGGATCCCAGCTCGATTTGGGTATCACCG-3′ | Outer primer #1 for introduction of hisGG233H−T235Q, BamHI site underlined |
hisG2 | 5′-CCAGTTGTCGCGTGCCAGTGGGGATACCTGTGGATGGGATAAGCCTGGGGTTACTGC-3′ | Inner primer #1 for introduction of hisGG233H−T235Q, affected triplets underlined |
hisG3 | 5′-GCAGTAACCCCAGGCTTATCCCATCCACAGGTATCCCCACTGGCACGCGACAACTGG-3′ | Inner primer #2 for introduction of hisGG233H−T235Q, affected triplets underlined |
hisG4 | 5′-CGCGGATCCGTTGATGGTGGTTCGTGAGATTTGG-3′ | Outer primer #2 for introduction of hisGG233H−T235Q, BamHI site underlined |
hisGseq | 5′-GGTATCCATCAAGCTTGG-3′ | Sequencing primer for hisGG233H−T235Q |
hisD1 | 5′-CCCAAGCTTCGGTGTCGCTGAAGTTAAGTTCTG-3′ | Outer primer #1 for exchange of native promoter with Ptuf in operon hisD–hisC–hisB–cg2302–cg2301, HindIII site underlined |
hisD2 | 5′-CGCAGGTCAGTGACATTCAACATATGTATTATGCATCTCGACCACCCCAGATTTACCTG-3′ | Inner primer #1 for exchange of native promoter with Ptuf in operon hisD–hisC–hisB–cg2302–cg2301, NdeI and NsiI sites underlined |
hisD3 | 5′-CAGGTAAATCTGGGGTGGTCGAGATGCATAATACATATGTTGAATGTCACTGACCTGCG-3′ | Inner primer #2 for exchange of native promoter with Ptuf in operon hisD–hisC–hisB–cg2302–cg2301, NdeI and NsiI sites underlined |
hisD4 | 5′-CGCGGATCCCTCATCAACACCCAAGATGGAAC-3′ | Outer primer #2 for exchange of native promoter with Ptuf in operon hisD–hisC–hisB–cg2302–cg2301, BamHI site underlined |
tuf1 | 5′-AACTGCAGAACCAATGCATCCACAGGGTAGCTGGTAGTTTG-3′ | Primer #1 for amplification of Ptuf, NsiI site underlined |
tuf2 | 5′-GGGAATTCCATATGATGTCCTCCTGGACTTCGTGGTGGC-3′ | Primer #2 for amplification of Ptuf, NdeI site underlined |
hisH1 | 5′-CCCAAGCTTCGGAGTGAAATGAGGTCCTTGGTC-3′ | Outer primer #1 for exchange of native promoter with Ptuf in operon hisH–hisA–impA–hisF–hisI–cg2294, HindIII site underlined |
hisH2 | 5′-GAGAAGGGCGACAGTTTTGGTCATATGTATTATGCATCTCCCTTCCTGAGAATGACGGCTAGTCG-3′ | Inner primer #1 for exchange of native promoter with Ptuf in operon hisH–hisA–impA–hisF–hisI–cg2294, NdeI and NsiI sites underlined |
hisH3 | 5′-CGACTAGCCGTCATTCTCAGGAAGGGAGATGCATAATACATATGACCAAAACTGTCGCCCTTCTC-3′ | Inner primer #1 for exchange of native promoter with Ptuf in operon hisH–hisA–impA–hisF–hisI–cg2294, NdeI and NsiI sites underlined |
hisH4 | 5′-CGCGGATCCGCACGAAGTAGAAACGCTCATCAGG-3′ | Inner primer #2 for exchange of native promoter with Ptuf in operon hisH–hisA–impA–hisF–hisI–cg2294, BamHI site underlined |
hisN1 | 5′-CCCAAGCTTCGCATGAGCATTTCAGCCCAGTCC-3′ | Outer primer #1 for exchange of native promoter with Ptuf in operon cg0911–hisN, HindIII site underlined |
hisN2 | 5′-GCTCTGGATTAGTCATGCCTTCCATATGTATTATGCATCTCGGTGGCGTTCTAAGAATCACAATCG-3′ | Inner primer #1 for exchange of native promoter with Ptuf in operon cg0911–hisN, NdeI and NsiI sites underlined |
hisN3 | 5′-CGATTGTGATTCTTAGAACGCCACCGAGATGCATAATACATATGGAAGGCATGACTAATCCAGAGC-3′ | Inner primer #2 for exchange of native promoter with Ptuf in operon cg0911–hisN, NdeI and NsiI sites underlined |
hisN4 | 5′-CGCGGATCCCACCGAACCACGTATAACCCATGG-3′ | Outer primer #2 for exchange of native promoter with Ptuf in operon cg0911–hisN, BamHI site underlined |
hisE1 | 5′-CCCAAGCTTCTGACAGAGTTGAAGGCCCTCGAG-3′ | Outer primer #1 for exchange of native promoter with PdapA–A16 and change of the translational start codon from GTG to ATG in operon hisE–hisG, HindIII site underlined |
hisE2 | 5′-CGTACAGCGAGTCAAATGTCTTCATATGTATTATGCATCTCGGAGGATATTAGTCGAATAATTTC-3′ | Inner primer #1 for exchange of native promoter with PdapA–A16 and change of the translational start codon from GTG to ATG in operon hisE–hisG, NdeI and NsiI sites underlined, base exchange bold |
hisE3 | 5′-GAAATTATTCGACTAATATCCTCCGAGATGCATAATACATATGAAGACATTTGACTCGCTGTACG-3′ | Inner primer #2 for exchange of native promoter with PdapA–A16 and change of the translational start codon from GTG to ATG in operon hisE–hisG, NdeI and NsiI sites underlined, base exchange bold |
hisE4 | 5′-CGCGGATCCGCAACGTAGATGGCGATATCTTTAGG-3′ | Outer primer #1 for exchange of native promoter with PdapA–A16 and change of the translational start codon from GTG to ATG in operon hisE–hisG, BamHI site underlined |
dapA1 | 5′-AACTGCAGAACCAATGCATTGGTTCTGCAGTTATCACACCC-3′ | Primer #1 for amplification of PdapA–A16, NsiI site underlined |
dapA2 | 5′-GGGAATTCCATATGAGGCTCCTTTTAAATCGAGCGGCTCCGGTCTTAGCTGTTAAACC-3′ | Primer #2 for amplification of PdapA–A16, NdeI site underlined |
hisF1 | 5′-CGCAAGCTTCTCCATGCCCATGCTGGGTAAACGC-3′ | Primer #1 for integration of stop codon and PsodA in front of hisF, HindIII site underlined |
hisF2 | 5′-GTCACAAGCCCGGAATAATTGGCAGTTATTTACTTGTACTCCTCATTTAACG-3′ | Primer #2 for integration of stop codon and PsodA in front of hisF, stop codon bold |
sodA1 | 5′-CGTTAAATGAGGAGTACAAGTAAATAACTGCCAATTATTCCGGGCTTGTGAC-3′ | Primer #3 for integration of stop codon and PsodA in front of hisF, stop codon bold |
sodA2 | 5′-GAATAACTCGAATTGCCACGCCCATGGGTAAAAAATCCTTTCGTAGGTTTC-3′ | Primer #4 for integration of stop codon and PsodA in front of hisF |
hisF3 | 5′-GAAACCTACGAAAGGATTTTTTACCCATGGGCGTGGCAATTCGAGTTATTC-3′ | Primer #5 for integration of stop codon and PsodA in front of hisF |
hisF4 | 5′-CGCGGATCCCGCCGCGCTTTGCCCACTCGATTGC-3′ | Primer #6 for integration of stop codon and PsodA in front of hisF, BamHI site underlined |
hisFseq | 5′-CTGACACCGAAGGCCATC-3′ |  |
tuf2_1 | 5′-GATCAGCGACGCCGCAGGGTCTAGACCACAGGGTAGCTGGTAGTTTGAAAATC-3′ | Primer #1 for amplification of Ptuf, XbaI site underlined |
tuf2_2 | 5′-CATTGAGCGCCGACAATAACGATTGCAGCCATTGTATGTCCTCCTGGACTTCG-3′ | Primer #2 for amplification of Ptuf |
purA1 | 5′-CGAAGTCCAGGAGGACATACAATGGCTGCAATCGTTATTGTCGGCGCTCAATG-3′ | Primer #1 for amplification of purA (cg3063) |
purA2 | 5′-GATCTTCTTTTTATCAGCCACTGTATGTCCTCCTGGACTTCCTAGTTGTCAGCTAGTACG-3′ | Primer #2 for amplification of purA (cg3063) |
purB1 | 5′-CGTACTAGCTGACAACTAGGAAGTCCAGGAGGACATACAGTGGCTGATAAAAAGAAGATC-3′ | Primer #1 for amplification of purB (cg2876) |
purB2 | 5′-CTTCTCTCATCCGCCAAAACAGTTAAAGAATCTCACCTGGTCGGTAGTC-3′ | Primer #2 for amplification of purB (cg2876) |
rrnB1 | 5′-GACTACCGACCAGGTGAGATTCTTTAACTGTTTTGGCGGATGAGAGAAG-3′ | Primer #1 for amplification of terminator region of rrnB (E. coli) |
rrnB2 | 5′-CTGCAGATATCCATCACACTGGCGGCCGCAGGAGAGCGTTCACCGACAAACAAC-3′ | Primer #2 for amplification of terminator region of rrnB (E. coli), NotI site underlined |
ABseq1 | 5′-GCGATTGAAGACCGTC-3′ | Sequencing primer for pJC4 purA purB |
ABseq2 | 5′-GCAATCGGCACCACCGGC-3′ | Sequencing primer for pJC4 purA purB |
ABseq3 | 5′-GCCTGCATGGGACGAAG-3′ | Sequencing primer for pJC4 purA purB |
ABseq4 | 5′-GTCACCGAACTGCTCAAC-3′ | Sequencing primer for pJC4 purA purB |
ABseq5 | 5′-GATGGAAGCAGGATCGCG-3′ | Sequencing primer for pJC4 purA purB |
pgi1 | 5′-CCCAAGCTTCAGCGTTGCGTGACGCACTCATTG-3′ | Outer primer #1 for introduction of pgiGTG, HindIII site underlined |
pgi2 | 5′-CCAAACCTGGGTGGTCGAAATGTCCGCCACGAAAACTCCTTTATTGTCG-3′ | Inner primer #1 for introduction of pgiGTG, nucleotide exchange bold |
pgi3 | 5′-CGACAATAAAGGAGTTTTCGTGGCGGACATTTCGACCACCCAGGTTTGG-3′ | Inner primer #2 for introduction of pgiGTG, nucleotide exchange bold |
pgi4 | 5′-CGCGGATCCCCAATGTTGACGATCTTCTTGATCG-3′ | Outer primer #2 for introduction of pgiGTG, BamHI site underlined |
pgiseq | 5′-CAAGCGTTGGGTTAAGGAGGA-3′ | Sequencing primer for pgiGTG |
gcv_Cjk_start_EcoRV | 5′-ATCGATATCCGAGAGGAGACACAACATGTCTTCTGCAGCTACTCGC-3′ | Primer #1 for amplification of gene cluster gcvPTH from C. jeikeium genome |
gcv_Cjk_MluI_XbaI | 5′-CAGTCTAGAACGCGTGGAACCGACCATAGGGTCTTG-3′ | Primer #2 for amplification of gene cluster gcvPTH from C. jeikeium genome |
lipB-Cjk_start-EcoRV | 5′-GCGGATATCATGGGATTCCAGCAAGGC-3′ | Primer #1 for amplification of gene cluster lipAB from C. jeikeium genome |
lipA-Cjk_stop-XbaI | 5′-GCGTCTAGATCCTTCGCCATGGATTCAAC-3′ | Primer #2 for amplification of gene cluster lipAB from C. jeikeium genome |
pK19-fw | 5′-TAATGCAGCTGGCACGAC-3′ | Primer #1 for sequencing the pK19mobsacB inserts |
pK19-rev | 5′-GTAGCTGACATTCATCCG-3′ | Primer #2 for sequencing the pK19mobsacB inserts |