TABLE 2.
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 |
Underlining represents the sequence of the restriction endonuclease identified in parentheses at the end of the sequence.