TABLE 3.
Primers used in this studya
| Entry | Primer name | Application/function | Sequence |
|---|---|---|---|
| 1 | SCO3334 (−380) For | Cloning of the trpRS1 leader | 5′-GATATCTTCTTTCGAACGTGGATTCCTC-3′b |
| 2 | SCO3334 (−1) Rev | Cloning of the trpRS1 leader | 5′-CATATGCTCTCCACCTCCTGGTCAG-3′c |
| 3 | SCO3334 (−59)A Rev | Cloning of the trpRS1 leader lacking part of the A+U-rich tract | 5′-CATATGCTCTCCACCTCCTTACGAGAACGGCCGCC-3′c,e |
| 4 | SCO3334 (−100)A Rev | Cloning of the trpRS1 leader lacking 3:4 hairpin | 5′-CATATGCTCTCCACCTCCTTGTGTACGGCCGCCGTC-3′c,e |
| 5 | TrpRS1 G(−126)A F | Site-directed mutagenesis of the trpRS1 leader, creation of G(−126)A mutation | 5′-GTACCCAGCTCTGGCAGGCCGCCTG-3′ |
| 6 | TrpRS1 G(−126)A R | Site-directed mutagenesis of the trpRS1 leader, creation of G(−126)A mutation | 5′-CGCCGTCAGGCGGCCTGCCAGAGCTG-3′ |
| 7 | TrpRS1 C(−127)A F | Site-directed mutagenesis of the trpRS1 leader, creation of C(−127)A mutation. | 5′-GTGTACCCAGCTCTGGAGGGCCGCCTG-3′ |
| 8 | TrpRS1 C(−127)A R | Site-directed mutagenesis of the trpRS1 leader, creation of C(−127)A mutation. | 5′-GCCGTCAGGCGGCCCTCCAGAGCTG-3′ |
| 9 | TrpRS1 G(−129)C F | Site-directed mutagenesis of the trpRS1 leader, creation of G(−129)C mutation | 5′-CCAGCTCTCGCGGGCCG-3′ |
| 10 | TrpRS1 G(−129)C R | Site-directed mutagenesis of the trpRS1 leader, creation of G(−129)C mutation. | 5′-CGGCCCGCGAGAGCTG-3′ |
| 11 | TrpRS1 C(−131)A F | Site-directed mutagenesis of the trpRS1 leader, creation of C(−131)A mutation | 5′-GTACGTGTACCCAGCTATGGCGGGCCGCCTG-3′ |
| 12 | TrpRS1 C(−131)A R | Site-directed mutagenesis of the trpRS1 leader, creation of C(−131)A mutation | 5′-GTCAGGCGGCCCGCCATAGCTGGGTAC-3′ |
| 13 | TrpRS1 T(−132)C F | Site-directed mutagenesis of the trpRS1 leader, creation of T(−132)C mutation | 5′-GTACGTGTACCCAGCCCTGGCGGGCCGCCTG-3′ |
| 14 | TrpRS1 T(−132)C R | Site-directed mutagenesis of the trpRS1 leader, creation of T(−132)C mutation | 5′-CAGGCGGCCCGCCAGGGCTGGGTAC-3′ |
| 15 | SAV4725 UF | Cloning of the SAV4725 leader | 5′- ACTAGTCGGAATCCTTGCTCCC-3′d |
| 16 | SAV4725 UR | Cloning of the SAV4725 leader | 5′-CATATGCTCTCCACCTCCTGGTCG-3′c |
| 17 | SAV4725 G(−125)C F1 | Site-directed mutagenesis of the SAV4725 leader, creation of G(−125)C mutation | 5′-GTACCCAGCAGTGGTCGGCCGCCTGAC-3′ |
| 18 | SAV4725 G(−125)C R1 | Site-directed mutagenesis of the SAV4725 leader, creation of G(−125)C mutation | 5′-CGCCGTCAGGCGGCCGACCACTGCTG-3′ |
| 19 | SAV4725 G(−128)C F1 | Site-directed mutagenesis of the SAV4725 leader, creation of G(−128)C mutation | 5′-CGTGTACCCAGCAGTCGTGGGCCGCC-3′ |
| 20 | SAV4725 G(−128)C R1 | Site-directed mutagenesis of the SAV4725 leader, creation of G(−128)C mutation | 5′-CGTCAGGCGGCCCACGACTGCTGGGTAC-3′ |
| 21 | SAV4725 RC A | SAV4725 transcript, 5′ RACE | 5′-CTGAAGATCCGCTTCATCTCTC-3′ |
| 22 | SAV4725 RC C | SAV4725 transcript, 5′ RACE | 5′-CCGTGCTGATCGACGTC-3′ |
| 23 | SAV4725 RC D | SAV4725 transcript, 5′ RACE | 5′-GTCGACGACGCAGAACAG-3′ |
a
Numbers in parentheses refer to positions relative to the predicted translation start site.
b
The engineered EcoRV site is underlined.
c
The engineered NdeI site is underlined.
d
The engineered SpeI site is underlined.
e
The predicted trpRS1 ribosome-binding site is in boldface.