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. 2020 Apr 1;86(8):e02757-19. doi: 10.1128/AEM.02757-19

TABLE 3.

Oligonucleotides used in this study

Oligonucleotide Sequence (5′–3′) Purpose and/or target
Gibson assembly
    1r-ST-Ptet-sadA-pWSK29 CCGGGCTGCAGGAATTCATGGCATTATGCCATTGC sadBA
    1f-ST_Ptet-fim-pWSK29 TCGACGGTATCGATAAGCTTAGGGAAAAAGGTTATGCTGCT sadBA
    Vf-pWSK29 GAATTCCTGCAGCCCGGGG Vector p4392
    Vr-pWSK29-Ptet-rev TTCACTTTTCTCTATCACTGATAGGGAGTGGTAAAATAACTCT Vector p4392
    1f-PtetA-hilD CCCTATCAGTGATAGAGAAAAGTGAAAACATCAACAAAGGGATAATATG hilD
    1r-hilD-vec CCCGGGCTGCAGGAATTCTGCCTGGCAGAAAGCTA hilD
    Vr-PtetA TTCACTTTTCTCTATCACTGATAGGGAGTGGTA Vector p4252
    hVPS26B-Rev-EcoRI TCAGAATTCTCTGCCTGCAGTTGTTGTCAGACAGC Vector pWSK29
    Vr-pWSK29 AAGCTTATCGATACCGTCGACCTC Vector pWSK29
    1f-FRT CGACGGTATCGATAAGCTTGAAAGTTCCTATACTTTCTAGAGAATA PtetA-misL
    1r-misL CCCGGGCTGCAGGAATTCCATGAAACCTATCAGCCAAA PtetA-misL
Site-directed mutagenesis
    ST-delbapA-Big1-Q5-fw CCTCTCCCCGATACACCG SDM p4253
    ST-delbapA-Big1-Q5-rv AGTATCTACAGGATTACTGCTACC SDM p4253
    ST-delbapA-Big28-Q5-fw ATAACCAGTCTTGATCTGAC SDM p4253
    ST-delbapA-Big28-Q5-rv CGTATCGACAATCACCGTC SDM p4253
    ST-delbapA-Big15-Q5-rv GGTGTCGACGGTGAAGGTAAAGCTG SDM p4253
    ST-delbapA-Big24-Q5-fw CTTGCGCCAACGGTTCCG SDM p4253
Check of P22 transductions and aph cassette removal
    K1-Red-Del CAGTCATAGCCGAATAGCCT aph
    K2-Red-Del CGGTGCCCTGAATGAACTGC aph
    CsgC-Check-rev TGTTGCCCTACCGCAGAATG csgBAC
    CsgG-Del-Check-for AGTGGGCTATGGCTGGCATC csgBAC-DEFG
    SPI4-Ctrl-For CGGTAGAGAATGGTCGGTAT SPI4
    SPI4-Ctrl-Rev GTGCTGACCTGATACGCTAT SPI4
    InvC-DelCheck-For TGTATCAGCGTCAAGGACGA invC
    InvC-DelCheck-Rev CGGCGAACAATAGACTGCTT invC
    SopB-DelCheck-Rev CAATGGCATAAAGGGACAGC sopB
    SopB-DelCheck-For TACGTATGGACGTCAGGATG sopB
    FLiI-DelCheck-For CGATCCAACGTTGCATCACG fliI
    FliI-DelCheck-Rev ACGCATTTCGCCCAGTAAAC fliI
    sopE2-check-rev4 GCGTCGCCATAAAAACGAATA sopE2
    SopE2-Red-Check-For TGTGACGCAGTAGTTGAATTGAAG sopE2
    sopA-DelCheck-Rev TTCGTACATGCGATGGTGAG sopA
    SopA-Check-For CCTGCCAGATAACATGGTGAATT sopA
    BapA-Check-For GTCAGGCACAAAAAACAAAGGGT bapABCD
    bapD-Check-Rev CCGAAATTCCTACATCCTCGG bapABCD
    STM3690-sadA-For GAGCATGGACAAACGTCACGC sadA
    SadA-end-Rev GGCATTATGCCATTGCCTTTG sadA
    ShdA-DelCheck-For GCCACAGCAAAGTTAAAGCG shdA
    ShdA-DelCheck-Rev TGAAGTCAAATCCGTCACGC shdA
    MisL-DelCheck-For TTTATGTGCATAAGCTGCGG misL
    MisL-DelCheck-Rev CAGGGCCATCGTGGCTTTAT misL
    SipA-Red-Check-For CACATTACAGACGCTGACGC sipA
    sopD-DelCheck-For ACCACAAAGGATTACCAACC sopD
    sopD-DelCheck-For GGCTGCATGAAGGGTAATTG sopD
    MotB-Check-Rev CCTGCAGAATAGTGAAGCCG motAB
    MotA-Check-For ATGAACAGATCGAACAGG motAB
    pagN-check-for CGTAGAAGTGAAACCGTACG pagN
    pagN-check-rev CAGCTATTTTACCGATAGTG pagN
    rck-check-for GAGGATGAAGCGGCGTTACG Rck
    rck-check-rev GTACCACACCACAAACCAGC Rck
    FliC-For-XhoI GCGCTCGAGGCAAACAGTAGTTAAGCGCG fliC
    FliC-Rev-EcoRI AGCGAATTCAGCTTTCGCTGCCTTGATTG fliC
    FljB-For-XhoI AGTCTCGAGCAATTTGCACTAGTAAGCGC fljB
    FljB-Rev GCAAGCATAGAATAATCCCG fljB
    CheZ-Check-For AAAACCATTCGCGCCGATAG cheZ
    CheZ-Check-Rev GGTAAAAAAGGCGGGGTTTAT cheZ
    CheY-DelCheck-Rev TACCGATGCGCGCAATGATG cheY
    CheY-DelCheck-For ACGAAGCAAGTTGTGTGGTG cheY
    Wzz-FepE-DelCheck-For AAACTATCGGGCCCATCATC fepE
    Wzz-FepE-DelCheck-Rev TGTTAAGCGATCTCAACCGC fepE
    WzzB-DelCheck-For AAAAGTGTATACCCGCGATC wzz
    WzzB-DelCheck-Rev AGTGATGTAGTGGCATTGAG wzz
    RfaL-DelCheck-For GCTGGCTGGCGCAAAATTTG rfaL
    RfaL-DelCheck-Rev TATTGTGCCATCTCAGGTTG rfaL
Red deletion
    CsgC-Red-del-rev CCGCCACCATCAAAAACTACTGTGCAGAAGGCGGCCATTGTGTAGGCTGGAGCTGCTTCG csgBAC
    CsgG-Red-Del-for CACGCTTTGTCGTATTCATCAGGATTCTGGCGGTACTGACATTCCGGGGATCCGTCGACC csgBAC-DEFG
    misL-Del13-For AGACGCTTTACGCCATAATGCAGGAGGCAGAATGCCAACTATTCCGGGGATCCGTCGACC misL
    misL-Del13-Rev ATCAGCGGCTCTGTTGTTACCTGAATCAGAAACTGTATTTTGTAGGCTGGAGCTGCTTCG misL
    ShdA-Del13-For AATAAAAGCAACGCGCGCGGCGCTGGCTTGCGCCGTGGCTATTCCGGGGATCCGTCGACC shdA
    ShdA-Del13-Rev GGCAGGGAACACCCGCCCGGTTTTGTCTAACTTACCAGTTTGTAGGCTGGAGCTGCTTCG shdA
    pagN-del-red-for GAAACTTGTCTTTTAGCCCAATATTAAGGCAGGTTCTGAAAGGGTTTTCCCAGTCACGAC pagN
    pagN-del-red-rev CATGAAGTCATTGGAGGCAGCCTTTGTGTCTGCATCATAATGCTTCCGGCTCGTATGTTG pagN
    rck-del-red-for CATAACACAATGAACTTAACTGTGTTCAGGGAGTTTTATCAGGGTTTTCCCAGTCACGAC rck
    rck-del-red-rev CGGAAGCCTGCGGCTCCGCTCCCTTTCCTGCTCTCCGTTATGCTTCCGGCTCGTATGTTG rck
    misL-Red-Ptet-For TTTTATAGATCCGTTTCCATTTTTATTATTTCCATATTATTGTAGGCTGGAGCTGCTTCG tetR PtetA
    misL-Red-Ptet-Rev ATGAGTAATTTTGGGGAGTTGGCATTCTGCCTCCTGCATTTTCACTTTTCTCTATCACTG tetR PtetA
    BapB-Del-Red-For GTTCGGGGCAACAAGCGGTGATATTTAAAAGGGATAAACTGTGTAGGCTGGAGCTGCTTC bapABCD
    BapD-Del-Red-Rev CACGCGTGACCAGCCCCCGTATCTTCTTATCTTCAACGATCATATGAATATCCTCCTTAG bapABCD
    SopA-Red-DEl13-For CCAGACCGTTTTTCCATAATGATGTTGATAAGGAATTCTAATTCCGGGGATCCGTCGAC sopA
    SopA-Red-Del13-Rev CAACGCTGTGTCCCTTAATTCCATGCGGGTTGAGGCTGGAGTAGGCTGGAGCTGCTTCG sopA
    sopD-Del13-For GATATTGAATAATATAAATTTGAAGGAAAATATTATGCCAATTCCGGGGATCCGTCGACC sopD
    sopD-Del13-rev CAGCCGGATTTTAAATTGGTTATATTACTGACTATCTTTATGTTGTAGGCTGGAGCTGCT sopD
Δ12 construction: PCR primers for cloning flanking regions
    100-LPF6-Bam TATCGGGGATCCGGGTTGAGTCGTATGACC lpf flanking region 1
    63-LPF5-Pst TATGCGCTGCAGGTGTATAGAGGTGGGTATTGG lpf flanking region 1
    64-LPF3-Pst TATCGCCTGCAGCATCTGGTGGGGAGCAACAATAC lpf flanking region 2
    101-LPF7-Bam TATCGGGGATCCGCCAAACAGTGAAAGAAGACGAAG lpf flanking region 2
    66-SAF1-Bam ATAGGCGGATCCCTGCACTGAAAAGCGATACC saf flanking region 1
    67-SAF2-Xba ATAGGCTCTAGAACGCCATACCAAATCTTACC saf flanking region 1
    92-SAF5-Xba TATCGCTCTAGACTGTTCCACTCATACTTCC saf flanking region 2
    69-SAF4-Bam TATGCGGGATCCTGGTCACAAGAAAGAGATGC saf flanking region 2
    70-STJ1-Bam TTACGCGGATCCCCTTTTTCGCCCATTACG stj flanking region 1
    71-STJ2-Xba TATCGGTCTAGAGGTCGGGATTCTATGAAG stj flanking region 1
    72-STJ3-Xba TATCGGTCTAGAGAAGTGCTGACGAAATAAACG stj flanking region 2
    73-STJ4-Bam ATACGCGGATCCGGCATGTTAGGTTTCACC stj flanking region 2
    78-STC5-Bam TTTGCGGGATCCAAGAGAATATGACATTCACTGC stc flanking region 1
    79-STC6-Xba ATAGCCTCTAGACATAGACAGGAAGTTATCGC stc flanking region 1
    80-STC7-Xba ATAGGCTCTAGACGATAGGTGAATGAACTTCC stc flanking region 2
    81-STC8-Sal TATGCCGTCGACCAGCAGAAATGATACACACG stc flanking region 2
    88-PEF5-Bam TTTGGCGGATCCTAATCTCACAGCCCGAAGC pef flanking region 1
    89-PEF6-Xba ATTGCCTCTAGACAGCTATGACGTGACATCG pef flanking region 1
    90-PEF7-Xba ATAGCGTCTAGAATGCGTGGTGTACTGAGG pef flanking region 2
    91-PEF8-Sal TAAGGGGTCGACGGCAGAAATGGTTTTGACG pef flanking region 2
Δ12 construction: PCR primers for confirmation of deletions
    34-KSAC-5out GGCATAAATTCCGTCAGC Amplify out KSAC 5′ end
    35-KSAC-3out TGATGACGAGCGTAATGG Amplify out KSAC 3′ end
    38-BCF-Up1 CATGATGACAAACGACTCC bcf deletions
    39-BCF-Down1 CGCCATTTGCAACATATCC bcf deletions
    40-FIM-Up1 CGTCTACGTCTTTATCTGG fim deletions
    41-FIM-Down1 GCACTTATCCTGTTGACC fim deletions
    42-LPF-Up1 GGGAGAATATCTGGAAAGC lpf deletions
    43-LPF-Down1 CAGCCACAATACAAAGTGC lpf deletions
    44-PEF-Up1 CGACAGGATATTTGCTCC pef deletions
    45-PEF-Down1 GTCAGTTTCCTTCATCACC pef deletions
    46-STB-Up1 ATATGTTCTCCCGAGTCG stb deletions
    47-STB-Down1 GTATGGCGGTATATTGTCG stb deletions
    48-STC-Up1 GGGGATATTCAGCTAACG stc deletions
    49-STC-Down1 GAGATCCAGGCAAAATCG stc deletions
    50-STD-Up1 TTCAGCAAACCCGTAAGG std deletions
    51-STD-Down1 GTGTAGCGATTCATCTGC std deletions
    52-STF-Up1 GCGTTTTACTGGTCTTTGC stf deletions
    53-STF-Down1 GTATCAACGGGAACTTTCG stf deletions
    54-STH-Up1 CCTTGTAGATGCCTATGC sth deletions
    55-STH-Down1 GGATTGGGACAACTTACC sth deletions
    56-STI-Up1 CAGAGACTGGTGACATCC sti deletions
    57-STI-Down1 AAGCTGAAATCGGAGACG sti deletions
    74-SAF-Up1 TATGATACCGAAGGAATACC saf deletions
    75-SAF-Down1 TCGACACGAAGCAAATCC saf deletions
    76-STJ-Up1 ACCCATGAACAGGTCTGC stj deletions
    77-STJ-Down1 ACTGAAGATGGCAACTCC stj deletions
Δ12 construction: PCR primers to check for presence of predicted major subunit
    145-bcfA1 GATACTACAACCGTCACT bcfA presence
    146-bcfA2 CCAACAGACGAGAAAAAAATCCCG bcfA presence
    147-fimA1 GCTGATCCTACTCCGGTG fimA presence
    148-fimA2 AAAATGGAACGCTGACGGGAGC fimA presence
    149-stbA1 GTTTCTGATAACACCATC stbA presence
    150-stbA2 GCTACCCAAAATAGTAACGCTCGC stbA presence
    151-stfA1 GCGGGCAGTAATACTGGT stfA presence
    152-stfA3 AGCCAGAACAATACCCACCACG stfA presence
    153-sthA1 TCCACACCGGTATTTGC sthA presence
    154-sth-II GGCATCAAGGCGAAAAAGAGG sthA presence
    155-stiA CAACAGGCAACAAAGCAACCC stiA presence
    156-stiC CCGCCAAAGACGGCACCG stiA presence
    157-safA1-Bam TTAGCGGGATCCGGCTCATTTTTGCCGAACTC safA presence
    158-safA2-Sal TTCACCGTCGACTTAAGGTTGATATCCCACTACG safA presence
    159-stjE1-Bam TTAGCGGGATCCGTTGAATCCACTGCTGTATTAAAACTG stjE presence
    160-stjE2-Sal TATGCCGTCGACCTGGTTGTAGCAAAGGAAGC stjE presence
    161-lpfA1 GCTGAATCTGGTGACGGC lpfA presence
    162-lpfA2 GATTCTCTTCCTGAGCCTCCG lpfA presence
    163-pefA1 GCCAATGAAGTAACTTTCCTGG pefA presence
    164-pefA2 GTTCTGCTTACGGGGGATTATTTG pefA presence
    165-stcA1 GTTGATGAGTATGATTCAGGC stcA presence
    166-stcA2 AACGACTTCTTTCTTCTCTGCCG stcA presence
    167-stdAF GCCGATACTACACCCACAGC stdA presence
    168-stdA2 CGACTTCAGGACGGAAAATGTC stdA presence