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. 2015 Jul 7;81(15):5157–5173. doi: 10.1128/AEM.00868-15

TABLE 2.

Primers used in this study

Primer purpose and primer DNA sequencea (5′–3′) Use
Gene disruption, complementation, and overexpression
    LWS1 CGGGATCCCCAGCGCCTTGACGGTCT (BamHI) Deletion of aveT gene
    LWS2 GTACTCGGCGGTGCTCGACTGCCTCCCACGCAGGAAT Deletion of aveT gene
    LWS3 ATTCCTGCGTGGGAGGCAGTCGAGCACCGCCGAGTAC Deletion of aveT gene
    LWS4 CCCAAGCTTTCGCTGTGCCCCTCCTTG (HindIII) Deletion of aveT gene
    LWS5 CCCAAGCTTGGAGGGAGGGGAGAGAGGG (HindIII) Overexpression of aveT in S. avermitilis
    LWS6 CGGGATCCCGAAGCAGGAGAGGCGAGTG (BamHI) Overexpression of aveT in S. avermitilis
    LWS7 GCGAGTGCGTCTTGAAGG Confirmation of aveT deletion in ΔaveT
    LWS8 CGGTGAGCAGCAGGGTCT Confirmation of aveT deletion in ΔaveT
    LWS9 CTACGACGCCCTGACCAT Confirmation of aveT deletion in ΔaveT
    LWS10 GCATCTCGTTCGTCTCGG Confirmation of aveT deletion in ΔaveT
    LWS11 CGGGATCCTCGGACTCGGGGTTCACCT (BamHI) Complementation of ΔaveT
    LWS12 GCTCTAGAGAGGGCGTACTCCCGGTC (XbaI) Complementation of ΔaveT
    LWS33 GGAATTCCGCTGAACGTGATCGTGCC (EcoRI) Deletion of aveM gene
    LWS34 GCTCTAGAAGGACGACCATCAACTGGG (XbaI) Deletion of aveM gene
    LWS35 GCTCTAGATACGGCGCTGCTGAACAC (XbaI) Deletion of aveM gene
    LWS36 CCCAAGCTTCGGAACCTCGCCTACGAC (HindIII) Deletion of aveM gene
    LWS37 AGGCAGACCTCCCATCCG Confirmation of aveM deletion in ΔaveM
    LWS38 TGCTCGACCTGCGCCTGA Confirmation of aveM deletion in ΔaveM
    LWS39 TTGTCGCTGAGCAACCAC Confirmation of aveM deletion in ΔaveM
    LWS40 GGAATACACCGAACATGCC Confirmation of aveM deletion in ΔaveM
    LWS41 GGAATTCAGGCAGACCTCCCATCCG (EcoRI) Complementation of ΔaveM
    LWS42 GCTCTAGAGCGCTCACATGTGGACGA (XbaI) Complementation of ΔaveM
    LWS43 GCTCTAGAGGCTTCCCTGGAGTGGTT (XbaI) Overexpression of aveM in S. avermitilis
    LWS44 CCCAAGCTTGCGCTCACATGTGGACGA (HindIII) Overexpression of aveM in S. avermitilis
    LWS52 GCTCTAGAGCGGAAAAGCATGGGTTAG (XbaI) Overexpression of pepD2 in S. avermitilis
    LWS53 CCCAAGCTTTCTGCCTTGTTCATCGTCT (HindIII) Overexpression of pepD2 in S. avermitilis
    LWS48 GGAATTCCATATGACTGAGACCGCAACGGTGCG (NdeI) Overexpression of His6-tagged AveT protein in E. coli
    LWS49 CGGGATCCTCAGGCGCCGAGGGCGGG (BamHI) Overexpression of His6-tagged AveT protein in E. coli
    GJ189 CGGAATTCTATCCCACCTCGTCGAACAC (EcoRI) Deletion of aco gene
    GJ190 GAAGATCTCGTAGCGATCATCGAGCTTC (BglII) Deletion of aco gene
    GJ191 GAAGATCTAGGAATGGCCACTGGTCTC (BglII) Deletion of aco gene
    GJ192 CCCAAGCTTGTTGCTCGCATGAGGTTCTT (HindIII) Deletion of aco gene
    GJ207 TCGACGTGAAGTGGAAGTAGAG Confirmation of aco deletion in Δaco
    GJ208 AGATGCAGGAACGCAGTACG Confirmation of aco deletion in Δaco
    aco-S GCGAGCATCCACTACAACCT Confirmation of aco deletion in Δaco
    aco-AS GGGGTCAGGAAGAGGAAGAC Confirmation of aco deletion in Δaco
5′ RACE
    Oligo(dT) anchor primer GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV
    Anchor primer GACCACGCGTATCGATGTCGAC
    aveTSP1 GCATCTCGTTCGTCTCGG Identification of TSS of aveT
    aveTSP2 CTCGCAGTTGTTCTCCCG Identification of TSS of aveT
    pepD2SP1 GGTGAAGTAGGAGAAGGGCT Identification of TSS of pepD2
    pepD2SP2 TGCTGCCCCAGTAGGTGAG Identification of TSS of pepD2
    pepD2SP3 CAGGTGGATCTCCGGGAC Identification of TSS of pepD2
    aveMSP1 CGTGTTCAGGAGCGAGAG Identification of TSS of aveM
    aveMSP2 CGGTGACCAGCATCTCGA Identification of TSS of aveM
    aveMSP3 AGAACCCGAGGTCCGCCT Identification of TSS of aveM
EMSA
    LWS13 TCGGACTCGGGGTTCACCT Probe aveT_pepD2_int
    LWS14 CTCGGGCGTGATCCGACT Probe aveT_pepD2_int
    LWS19 GTGACCCACGACACGTACGAAACGGTTTCGTTTCGCTCGCTCT Probe 1
    LWS20 AGAGCGAGCGAAACGAAACCGTTTCGTACGTGTCGTGGGTCAC Probe 1
    LWS21 GTGACCCACGACACGTAAAGCTTGGTTTGGAATTCCTCGCTCT Probe 1m
    LWS22 AGAGCGAGGAATTCCAAACCAAGCTTTACGTGTCGTGGGTCAC Probe 1m
    LWS23 TGGTGCCTCGGTCCTTGG Probe aveM_sav_7491_int
    LWS24 CGGGTGCTTTCGGTCAGA Probe aveM_sav_7491_int
    LWS50 ACGCCTGGTCCTCCGA Probe aveRp
    LWS51 TGAGTTCTTCTGGTTTCCGAG Probe aveRp
    LWS58 ATGGTCGGGAACCTCCGCAA Probe aveA1p
    LWS59 CTGTGTCCTCACCGCTAGGC Probe aveA1p
DNase I footprinting assay
    FAM-LWS15 CCAGCCACAGGTCGTCCT aveT-pepD2 intergenic region
    LWS16 TGGTCAGGGCGTCGTAGC aveT-pepD2 intergenic region
Real-time RT-PCR
    LWS25 CCGTGTCGTTCGAAGCA aveT ORF
    LWS26 GAGTACAGCTCGGCCTC aveT ORF
    LWS27 CAAGGCGAAGAAGTCCGAAC pepD2 ORF
    LWS28 CCGCAGATCTCCTTCGTCCA pepD2 ORF
    LWS29 GCGACCGGCTATCTGTCC aveM ORF
    LWS30 GAAGAAGACCGCCGACCAC aveM ORF
    LWS31 ACGCTCACCAACGTCCT sav_7491 ORF
    LWS32 CCCGCCTCGACGTAGCC sav_7491 ORF
    LWS54 CAGAAGAACTCACGCTCGTC aveR ORF
    LWS55 ACTCTTTCCACAGCCCATTC aveR ORF
    LWS56 CGGACAGGACTACGCACTTC aveA1 ORF
    LWS57 ACGAGATACGACCGGAGATG aveA1 ORF
a

Underlining represents the sequence of the restriction endonuclease identified in parentheses at the end of the sequence.