Table 2.
Plasmids used and generated in this study
| Plasmid | Description | Resistance phenotypea | Reference |
|---|---|---|---|
| pRMH977 | Single IS26 cloned into pUC19b | Ap | (27) |
| pRMH1016 | pRMH977 with 7G>T transversion in TIRR | Ap | This study |
| pRMH1017 | pRMH977 with 10G>T transversion in TIRR | Ap | This study |
| pRMH1003 | pRMH977 with transversions of bp 1–2 of the IS26 TIRR | Ap | (31) |
| pRMH1018 | pRMH977 with transversions of bp 3–5 of the IS26 TIRR | Ap | This study |
| pRMH1019 | pRMH977 with transversions of bp 6–8 of the IS26 TIRR | Ap | This study |
| pRMH1020 | pRMH977 with transversions of bp 9–11 of the IS26 TIRR | Ap | This study |
| pRMH1021 | pRMH977 with transversions of bp 12–14 of the IS26 TIRR | Ap | This study |
| pRMH977-F4A | pRMH977 encoding Tnp26 F4A | Ap | This study |
| pRMH977-F9A | pRMH977 encoding Tnp26 F9A | Ap | This study |
| pRMH977-F4AF9A | pRMH977 encoding Tnp26 F4AF9A | Ap | This study |
| pRMH977-E30P | pRMH977 encoding Tnp26 E30P | Ap | This study |
| pRMH977-E30PW50P | pRMH977 encoding Tnp26 E30PW50P | Ap | This study |
| pRMH977-G39I | pRMH977 encoding Tnp26 G39I | Ap | This study |
| pRMH977-G39W | pRMH977 encoding Tnp26 G39W | Ap | This study |
| pRMH977-R49A | pRMH977 encoding Tnp26 R49A | Ap | This study |
| pRMH977-W50A | pRMH977 encoding Tnp26 W50A | Ap | This study |
| pRMH977-W50P | pRMH977 encoding Tnp26 W50P | Ap | This study |
| pRMH977-Y54A | pRMH977 encoding Tnp26 Y54A | Ap | This study |
| R388 | Conjugative IncW plasmid | SuTp | (43) |
| R388::IS26 | R388 with IS26c | SuTp | (27) |
| R388::IS26-E30P | R388::IS26 encoding Tnp26 E30P | SuTp | This study |
| R388::IS26-E30PW50P | R388::IS26 encoding Tnp26 E30PW50P | SuTp | This study |
| R388::IS26-G39I | R388::IS26 encoding Tnp26 G39I | SuTp | This study |
| R388::IS26-G39W | R388::IS26 encoding Tnp26 G39W | SuTp | This study |
| R388::IS26-R49A | R388::IS26 encoding Tnp26 R49A | SuTp | This study |
| R388::IS26-W50A | R388::IS26 encoding Tnp26 W50A | SuTp | This study |
| R388::IS26-W50P | R388::IS26 encoding Tnp26 W50P | SuTp | This study |
| R388::IS26-Y54A | R388::IS26 encoding Tnp26 Y54A | SuTp | This study |
| 1C | E. coli expression vector carrying an ORF encoding N-terminal His6-MBP-Asn10-TEVd | Km | Scott Gradiae |
| 1C::Tnp261–56 | 1C encoding Tnp26 M1–P56 | Km | This study |
| 1C::Tnp261–71 | 1C encoding Tnp26 M1–P71 | Km | This study |
| 1C::Tnp261–234 | 1C encoding Tnp26 M1–M234 | Km | This study |
| 1C::Tnp261–234 F4A | 1C encoding Tnp26 M1–M234 F4A | Km | This study |
| 1C::Tnp261–234 F9A | 1C encoding Tnp26 M1–M234 F9A | Km | This study |
| 1C::Tnp261–234 R49A | 1C encoding Tnp26 M1–M234 R49A | Km | This study |
| 1C::Tnp261–234 W50A | 1C encoding Tnp26 M1–M234 W50A | Km | This study |
| 1C::Tnp261–234 Y54A | 1C encoding Tnp26 M1–M234 Y54A | Km | This study |
a
Ap, ampicillin; Km, kanamycin; Su: sulfamethoxazole; and Tp, trimethoprim.
b
IS26 together with bases 119,362 to 119,454 and 122,137 to 122,225 from GenBank accession number KF976462. Cloned the insert with tnp26 facing toward Plac in pUC19.
c
IS26 8-bp duplication of bases 26,745 to 26,752 in R388 (GenBank accession no.: BR000038).
d
MBP, maltose-binding protein; TEV, tobacco etch virus cleavage site.
e
pET His6 MBP Asn10 TEV LIC cloning vector (1C) was a gift from Scott Gradia (Addgene plasmid #29654).