TABLE 1.
Primers used in this study
| Primer | Description | Sequence |
|---|---|---|
| tdk-Kanr gene cassette | ||
| P1-F | tdk-Kanr gene amplification for deletion and rescue cassettes | CCCAGCCTCCAATTCAAATCA |
| P2-R | tdk-Kanr gene amplification for deletion and rescue cassettes | CCAGCTCCGCATGCTTAGAA |
| tdk-R | Used with P1-F to amplify just the tdk gene from the tdk-KanR gene cassette | ACATCAGAGATTTTGAGACACAACGTG |
| Deletion of IS1236 elements to create ISx-ADP1 | ||
| IS#1-P3-F | Amplifies 5′ flank for IS1236 no. 1 deletion and rescue cassettes | GCTTGAACCTCACTCCTGA |
| IS#1-P1′P4-R | Amplifies 5′ flank with IS#1-P3-F for IS1236 no. 1 deletion cassette | TGATTTGAATTGGAGGCTGGGACAATTGCATAAAAATGAAAGTAACTC |
| IS#1-P2′P5-F | Amplifies 3′ flank with IS#1-P6-R for IS1236 no. 1 deletion cassette | TTCTAAGCATGCGGAGCTGGTGTCCAGTCAATTGCTTTAATG |
| IS#1-P6-R | Amplifies 3′ flank for IS1236 no. 1 deletion and rescue cassettes | TCGATAGACTAAAATGACTAGACAGG |
| IS#1-P5′P4-R | Amplifies 5′ flank with IS#1-P3-F for IS1236 no. 1 deletion cassette | CATTAAAGCAATTGACTGGACAACAATTGCATAAAAATGAAAGTAACTC |
| IS#1-P4′P5-F | Amplifies 3′ flank with IS#1-P6-R for IS1236 no. 1 deletion cassette | GAGTTACTTTCATTTTTATGCAATTGTTGTCCAGTCAATTGCTTTAATG |
| IS#23-P3-F | Amplifies 5′ flank for Tn5613 deletion and rescue cassettes | TAAAGGACCTGATAAGGCGAT |
| IS#23-P1′P4-R | Amplifies 5′ flank with IS#23-P3-F for Tn5613 deletion cassette | TGATTTGAATTGGAGGCTGGGCTTTAATGATTAAATAATCAATTTTCATCT |
| IS#23-P2′P5-F | Amplifies 3′ flank with IS#23-P6-R for Tn5613 deletion cassette | TTCTAAGCATGCGGAGCTGGTCAATTGACCGCTTGATCA |
| IS#23-P6-R | Amplifies 3′ flank for Tn5613 deletion and rescue cassettes | GATCTATTTGGCAATATCATAAACATC |
| IS#23-P5′P4-R | Amplifies 5′ flank with IS#23-P3-F for Tn5613 deletion cassette | TGATCAAGCGGTCAATTGACTTTAATGATTAAATAATCAATTTTCATCT |
| IS#23-P4′P5-F | Amplifies 3′ flank with IS#23-P6-R for Tn5613 deletion cassette | AGATGAAAATTGATTATTTAATCATTAAAGTCAATTGACCGCTTGATCA |
| IS#4-P3-F | Amplifies 5′ flank for IS1236 no. 4 deletion and rescue cassettes | GGCGATTAAGAGAGTAATTCCA |
| IS#4-P1′P4-R | Amplifies 5′ flank with IS#4-P3-F for IS1236 no. 4 deletion cassette | TGATTTGAATTGGAGGCTGGGATGAACATCTTCAACATTTAGATCAAG |
| IS#4-P2′P5-F | Amplifies 3′ flank with IS#4-P6-R for IS1236 no. 4 deletion cassette | TTCTAAGCATGCGGAGCTGGCATGCATTGCACGTTCG |
| IS#4-P6-R | Amplifies 3′ flank for IS1236 no. 4 deletion and rescue cassettes | AATGCCAGATCTACACTGACG |
| IS#4-P5′P4-R | Amplifies 5′ flank with IS#4-P3-F for IS1236 no. 4 deletion cassette | CGAACGTGCAATGCATGATGAACATCTTCAACATTTAGATCAAG |
| IS#4-P4′P5-F | Amplifies 3′ flank with IS#4-P6-R for IS1236 no. 4 deletion cassette | CTTGATCTAAATGTTGAAGATGTTCATCATGCATTGCACGTTCG |
| IS#5-P3-F | Amplifies 5′ flank for IS1236 no. 5 deletion and rescue cassettes | GAATTTAAAGTTAAAGATATTTTTGCG |
| IS#5-P1′P4-R | Amplifies 5′ flank with IS#5-P3-F for IS1236 no. 5 deletion cassette | TGATTTGAATTGGAGGCTGGGGTTTGAGTTTATCGGAAAAACTATC |
| IS#5-P2′P5-F | Amplifies 3′ flank with IS#5-P6-R for IS1236 no. 5 deletion cassette | TTCTAAGCATGCGGAGCTGGAACAAAAGGCCATGGAG |
| IS#5-P6-R | Amplifies 3′ flank for IS1236 no. 5 deletion and rescue cassettes | GTACCTTGCCGAGCAAA |
| IS#5-P5′P4-R | Amplifies 5′ flank with IS#5-P3-F for IS1236 no. 5 deletion cassette | CTCCATGGCCTTTTGTTGTTTGAGTTTATCGGAAAAACTATC |
| IS#5-P4′P5-F | Amplifies 3′ flank with IS#5-P6-R for IS1236 no. 5 deletion cassette | GATAGTTTTTCCGATAAACTCAAACAACAAAAGGCCATGGAG |
| IS#6-P3-F | Amplifies 5′ flank for IS1236 no. 6 deletion and rescue cassettes | GGAGTATCGTGATCCCAAAG |
| IS#6-P1′P4-R | Amplifies 5′ flank with IS#6-P3-F for IS1236 no. 6 deletion cassette | TGATTTGAATTGGAGGCTGGGCTACTCAGTTAGTCGAAAGTATCATCC |
| IS#6-P2′P5-F | Amplifies 3′ flank with IS#6-P6-R for IS1236 no. 6 deletion cassette | TTCTAAGCATGCGGAGCTGGGTTCAAGTGGGGTTTACTGG |
| IS#6-P6-R | Amplifies 3′ flank for IS1236 no. 6 deletion and rescue cassettes | CAGTGATATGCATGCTGAGG |
| IS#6-P5′P4-R | Amplifies 5′ flank with IS#6-P3-F for IS1236 no. 6 deletion cassette | CCAGTAAACCCCACTTGAACCTACTCAGTTAGTCGAAAGTATCATCC |
| IS#6-P4′P5-F | Amplifies 3′ flank with IS#6-P6-R for IS1236 no. 6 deletion cassette | GGATGATACTTTCGACTAACTGAGTAGGTTCAAGTGGGGTTTACTGG |
| qPCR to monitor IS1236 deletion | ||
| qIS-F | Amplifies 119-bp fragment common to all IS1236 elements with qIS-R | GTTTTCGCCAGGCATAATA |
| qIS-R | Amplifies 119-bp fragment common to all IS1236 elements with qIS-F | CAGATCATGCCAAGAAAAGTAC |
| Repair of rpoD and cyoB mutations | ||
| rpoD-F | Amplifies fragment containing rpoD mutation with rpoD-R | GCCTTCAAACAATTCACTTAACC |
| rpoD-R | Amplifies fragment containing rpoD mutation with rpoD-F | GCAATCGAAATAACGAGACG |
| rpoD-S | Sequence mutation in rpoD gene | GCTGAGGTTAACGATCATCT |
| cyoB-F | Amplifies fragment containing cyoB mutation with cyoB-R | GGTACGCTTTTATCTGGTGTAA |
| cyoB-R | Amplifies fragment containing cyoB mutation with cyoB-F | CAGCAGAATTATGATCATGACTC |
| cyoB-S | Sequence mutation in cyoB gene | GACACGTCGTTTGAATACAT |
| Integration of tdk-Kanr gene cassette for measuring inactivating mutation rates and transformation frequency | ||
| Site1-P3-F | Amplifies 5′ flank for integration of tdk-Kanr gene cassette | ATAAAAAATTTTTTATTATAATTAATAATTAAGTTGTG |
| Site1-P1′P4-R | Amplifies 5′ flank with Site1-P3-F for integration of tdk-Kanr gene cassette | TGATTTGAATTGGAGGCTGGGATCTAAACCTTGTGATATCAACTGTT |
| Site1-P2′P5-F | Amplifies 3′ flank with Site1-P6-R for integration of tdk-Kanr gene cassette | TTCTAAGCATGCGGAGCTGGGTGCAAAGAATATTCATCTGAATG |
| Site1-P6-R | Amplifies 3′ flank for integration of tdk-Kanr gene cassette | TACTTCAGATTCTGGGAATTTCT |
| Site2-P3-F | Amplifies 5′ flank for integration of tdk-Kanr gene cassette | CAAATCCATTGAAAAGAAATGC |
| Site2-P1′P4-R | Amplifies 5′ flank with Site2-P3-F for integration of tdk-Kanr gene cassette | TGATTTGAATTGGAGGCTGGGCTGATCATCATGGACAAGCT |
| Site2-P2′P5-F | Amplifies 3′ flank with Site2-P6-R for integration of tdk-Kanr gene cassette | TTCTAAGCATGCGGAGCTGGTGTGATCTTTTTTTCAAATTCATT |
| Site2-P6-R | Amplifies 3′ flank for integration of tdk-Kanr gene cassette | GGTAATGTTCTCTCCTACGAAAAG |
| Site3-P3-F | Amplifies 5′ flank for integration of tdk-Kanr gene cassette | CCTGATCCAGAAAGATATATGACTT |
| Site3-P1′P4-R | Amplifies 5′ flank with Site3-P3-F for integration of tdk-Kanr gene cassette | TGATTTGAATTGGAGGCTGGGTGAAAAGTTATCAAATATTGACGTATG |
| Site3-P2′P5-F | Amplifies 3′ flank with Site3-P6-R for integration of tdk-Kanr gene cassette | TTCTAAGCATGCGGAGCTGGTAAAGTGATCGAGCAGCCA |
| Site3-P6-R | Amplifies 3′ flank for integration of tdk-Kanr gene cassette | AACTATACATATTGAATTTTTAAATAATAATTTAATTC |
| Integration of Specr genes in region 1 for measuring point mutation rates and transformation frequency | ||
| R1-P3-F | Amplifies R1 5′ flank for integration of tdk-Kanr gene cassette | ACGCCGAGTCCTCTTGAGTACAGG |
| R1-P1′P4-R | Amplifies R1 5′ flank with Site1-P3-F for integration of tdk-Kanr gene cassette | TGATTTGAATTGGAGGCTGGGGATTTTCCGCCCATCTCAC |
| R1-P2′P5-F | Amplifies R1 3′ flank with Site1-P6-R for integration of tdk-Kanr gene cassette | TTCTAAGCATGCGGAGCTGGCAGAAATTATAAAACGCACATCA |
| R1-P6-R | Amplifies R1 3′ flank for integration of tdk-Kanr gene cassette | ACTCGCTGCAATAGTGGCAAAAGC |
| Specr-F | Amplifies Specr gene to construct rescue cassette | TTCAAATATGTATCCGCTCATGAGACAATAAC |
| Specr-R | Amplifies Specr gene to construct rescue cassette | TTATTTGCCGACTACCTTGGTGATC |
| Specr-E8*-R | Amplifies 5′ end of Specr gene with Specr-F to introduce stop codon | CTGATAGTTGAGTCGATACTTAGGCGA |
| Specr-E8*-F | Amplifies 3′ end of Specr gene with Specr-R to introduce stop codon | GCCTAAGTATCGACTCAACTATCAGAGGTA |
| Specr-R1-R | Amplifies R1 5′ flank with R1-P3-F to create overlap with Specr gene | ATGAGCGGATACATATTTGAAGATTTTCCGCCCATCTCAC |
| Specr-R1-F | Amplifies R1 3′ flank with R1-P6-R to create overlap with Specr gene | ACCAAGGTAGTCGGCAAATAATCTTTACGAGTCATGCCAGCAC |
| Deletion of dinP and umuDAb | ||
| dinP-P3-F | Amplifies 5′ flank for dinP deletion and rescue cassettes | CAAAGCGATAATGTAGAAAAAAC |
| dinP-P1′P4-R | Amplifies 5′ flank with dinP-P3-F for dinP deletion cassette | TGATTTGAATTGGAGGCTGGGTCGTATTTTTTTTACACAAAAA |
| dinP-P2′P5-F | Amplifies 3′ flank with dinP-P6-R for dinP deletion cassette | TTCTAAGCATGCGGAGCTGGTCATTTTACTGTATTTGTGATTT |
| dinP-P6-R | Amplifies 3′ flank for IS1236 no. 1 deletion and rescue cassettes | ATGCTCTCGATGAGTATTGG |
| dinP-P5′P4-R | Amplifies 5′ flank with dinP-P3-F for dinP deletion cassette | CTCAAATCACAAATACAGTAAAATGATCGTATTTTTTTTACACAAAAATTTAA |
| dinP-P4′P5-F | Amplifies 3′ flank with dinP-P6-R for dinP deletion cassette | TTAAATTTTTGTGTAAAAAAAATACGATCATTTTACTGTATTTGTGATTTGAG |
| umuD-P3-F | Amplifies 5′ flank for umuD deletion and rescue cassettes | TTGGCTCCACTACTCACAGA |
| umuD-P1′P4-R | Amplifies 5′ flank with umuD-P3-F for umuD deletion cassette | TGATTTGAATTGGAGGCTGGGTCATGAGTCAGAGAATCTTTGC |
| umuD-P2′P5-F | Amplifies 3′ flank with umuD-P6-R for umuD deletion cassette | TTCTAAGCATGCGGAGCTGGAATGTTTTCTCAAGTTAAAATAATCTAA |
| umuD-P6-R | Amplifies 3′ flank for umuD deletion and rescue cassettes | ATCTATACTAGTAGATTATACGGACGATG |
| umuD-P5′P4-R | Amplifies 5′ flank with umuD-P3-F for umuD deletion cassette | TTAGATTATTTTAACTTGAGAAAACATTTCATGAGTCAGAGAATCTTTGC |
| umuD-P4′P5-F | Amplifies 3′ flank with umuD-P6-R for umuD deletion cassette | GCAAAGATTCTCTGACTCATGAAATGTTTTCTCAAGTTAAAATAATCTAA |