TABLE 3.
Primers used in this study
| Entry | Primer name | Application or function | Sequence (reference) |
|---|---|---|---|
| 1 | TrpRS1 MK54 | Detection of the S. coelicolor trpRS1 (SCO3334) transcript via RT-PCR | 5′-CAG GAG GTG GAC ATA TGA CAC GGG TTT TCA G-3′ (22) |
| 2 | TrpRS1 MK134 | Detection of the S. coelicolor trpRS1 (SCO3334) transcript via RT-PCR | 5′-CAC GTA CCC GGG ATC GGC GGA CAG C-3′ (22) |
| 3 | TrpRS2 MK133 | Detection of the S. coelicolor trpRS2 (SCO4839) transcript via RT-PCR | 5′-GCG TCT CCG GGT CGT CCA GGT ACT G-3′ (22) |
| 4 | TrpRS2 MK135 | Detection of the S. coelicolor trpRS2 (SCO4839) transcript via RT-PCR | 5′-CGC GAC CTC GCG GAC CGC TTC AAC CAG-3′ (22) |
| 5 | SAV3417 RT FOR | Detection of the S. avermitilis SAV3417 transcript via RT-PCR | 5′-CCA CCT CGG CAA CTA CCT C-3′ |
| 6 | SAV3417 RT REV | Detection of the S. avermitilis SAV3417 transcript via RT-PCR | 5′-TTG CCG GTG TAC TTC TCC TC-3′ |
| 7 | SAV4725 RT FOR | Detection of the S. avermitilis SAV4725 transcript via RT-PCR | 5′-ATC TGA CGC TGG GGA ACT AC-3′ |
| 8 | SAV4725 RT REV | Detection of the S. avermitilis SAV4725 transcript via RT-PCR | 5′-CCG TAC CGC TGG TTG AAG-3′ |
| 9 | SGR2702 RT FOR | Detection of the S. griseus NBRC 13350 SGR2702 transcript and corresponding transcript from the indolmycin producer S. griseus ATCC 12648 via RT-PCR | 5′-ACG ACG CCT TCT ACA TGG TC-3′ |
| 10 | SGR2702 RT REV | Detection of the S. griseus NBRC 13350 SGR2702 transcript and corresponding transcript from the indolmycin producer S. griseus ATCC 12648 via RT-PCR | 5′-GCC TGG TAG AGC AGG ATG TC-3′ |
| 11 | SGR3809 RT FOR 1 | Detection of the S. griseus NBRC 13350 SGR3809 transcript via RT-PCR | 5′-ACA CCC GCC GTC ACC AC-3′ |
| 12 | SGR3809 RT REV 1 | Detection of the S. griseus NBRC 13350 SGR3809 transcript via RT-PCR | 5′-GAG AGC ATG ATG TCC GGT TC-3′ |
| 13 | SGR3809 RT FOR 2 | Detection of the S. griseus ATCC 12648 SGR3809 homolog transcript via RT-PCR | 5′-GGC TGC TCC TGG ACT ATC TG-3′ |
| 14 | SGR3809 RT REV 2 | Detection of the S. griseus ATCC 12648 SGR3809 homolog transcript via RT-PCR | 5′-GTC GGG GTC GTA GGT GAT G-3′ |
| 15 | SCO4839 KO DET FOR | Cloning of the S. coelicolor trpRS2 locus located 93 nucleotides upstream of the translational start site | 5′-CCC CGC CCT TGT TAC AC-3′ |
| 16 | SCO4839 KO DET REV | Cloning of the S. coelicolor trpRS2 locus located 96 nucleotides downstream of the translational stop site | 5′-CAA CCG GAG TGA TGT GCA G-3′ |
| 17 | SAV4725 FOR | Cloning of the S. avermitilis SAV4725 ORF | 5′-CAT ATG AAG CGG ATC TTC AGC-3′a |
| 18 | SAV4725 REV | Cloning of the S. avermitilis SAV4725 ORF | 5′-CTC GAG TTA CGC CTC CAG CAA CC-3′b |
| 19 | SCO3334 FOR | Cloning of the S. coelicolor trpRS1 ORF | 5′-CAT ATG ACA CGG GTT TTC AGT G-3′a |
| 20 | SCO3334 REV | Cloning of the S. coelicolor trpRS1 ORF | 5′-CTC GAG CTA CCG GGC CGC GTT C-3′b |
| 21 | SGR3809 FOR | Cloning of the S. griseus NBRC 13350 SGR3809 ORF | 5′-CAT ATG ACC ACA CCC GCC G-3′a |
| 22 | SGR3809 REV 2.0 | Cloning of the S. griseus NBRC 13350 SGR3809 ORF | 5′-CTC GAG CTA CCG GAA CGC GCC CAT C-3′b |
| 23 | SCO4839 L9F FOR | Site-directed mutagenesis of the S. coelicolor trpRS2 locus, creation of L9F mutation | 5′-GAA CGT CCC CGT GTG TTC TCC GGT ATC CAG C-3′ |
| 24 | SCO4839 L9F REV | Site-directed mutagenesis of the S. coelicolor trpRS2 locus, creation of L9F mutation | 5′-GCT GGA TAC CGG AGA ACA CAC GGG GAC-3′ |
| 25 | SCO4839 Q13K FOR | Site-directed mutagenesis of the S. coelicolor trpRS2 locus, creation of Q13K mutation | 5′-GTG CTC TCC GGT ATC AAG CCG ACC TC-3′ |
| 26 | SCO4839 Q13K REV | Site-directed mutagenesis of the S. coelicolor trpRS2 locus, creation of Q13K mutation | 5′-GAG CCG GAG GTC GGC TTG ATA CCG GAG-3′ |
| 27 | SCO4839 H48Q FOR | Site-directed mutagenesis of the S. coelicolor trpRS2 locus, creation of H48Q mutation | 5′-GGT CGT CGA CCT GCA GGC GAT CAC GG-3′ |
| 28 | SCO4839 H48Q REV | Site-directed mutagenesis of the S. coelicolor trpRS2 locus, creation of H48Q mutation | 5′-CGG GAC CGT GAT CGC CTG CAG GTC G-3′ |
a
The engineered NdeI site is underlined.
b
The engineered XhoI site is underlined.