TABLE 2.
List of primers used in this work
| Name | Sequence 5′−3′ | Description |
|---|---|---|
| Confirm pCasPA in Burkholderia | ||
| confCas9_F confCas9_R |
GGAAGCGACTCGTCTCAAAC TTCTTCTTGGCTAGCTCCCC |
Amplification of a 937-bp internal fragment from the beginning of cas9 gene present in plasmid pCasPA Amplification of a 936-bp internal fragment from the end of cas9 gene present in plasmid pCasPA |
| confCas9_2_F confCas9_2_R |
AGCTGAACGTGGAGGTTTGA
AGCATCCGTTTACGACCGTT |
|
| Exchange of selection marker in pACRISPR | ||
| cat_bbsI_F | CGAAGACGAAAGGTCGAATAAATACCTGTGACGGA | Amplification of cat coding sequence and promoter region from pBBR1MCS |
| cat_ageI_R | CCACCGGTGCTGCATTAATGAATCGGCC | |
| Km_pACRISPR_F | GAAGACGAAAGGATGTCAGCTACTGGGCTATC | Amplification of aminoglycoside 3′-phosphotransferase coding sequence and promoter region from pK19mob |
| Km_pACRISPR_R | ACCGGTGTCATTTCGAACCCCAGAGTC | |
| dhfr_bbsI_F | GAAGACGAAAGGCCGTGGGTCGATGTTTGATG | Amplification of dhfr coding sequence and promoter region from pUC-TP |
| dhfr_ageI_R | ACCGGTCTGGAGCGAATTGTTAGGCC | |
| Test spacer presence | ||
| test_spacer_F | GGATTTGCAGACTACGGGCCTA | Amplification of the region where spacer insertion occurs |
| test_spacer_R | CGACTCGGTGCCACTTTTTCAA | |
| Deletion of the rpfR gene in P0213-1 | ||
| rpfR_spacer_F | GTGGGGTATCGGTGTACGCCGACG | rpfR spacer for gene deletion in P0213-1 |
| rpfR_spacer_R | AAACCGTCGGCGTACACCGATACC | |
| rpfR_P1 | TTTTGAGATCTGTCCATACCCATGGTCTAGATGCAGACGCACATGACGAA | Amplification of 779 bp region upstream rpfR |
| rpfR_P2 | GACAGCCGGTTCGGAACGCCGCGAATGACGGACCCATCTT | |
| rpfR_P3 | AAGATGGGTCCGTCATTCGCGGCGTTCCGAACCGGCTGTC | Amplification of 620 bp region downstream rpfR |
| rpfR_P4 | TCTGAATGGCGGGAGTATGAAAAGTCTCGAGGGCGCGATCTGCTGATGGCAT | |
| conf_rpfR_F | CAGGGGACGATGAACAACTT | Amplification of 1.663 bp to confirm the mutant (3.846 bp in the wild type) |
| conf_rpfR_R | CTTCAACCAGCAACTCGTCA | |
| conf_rpfR_final_F | AGCATTCCGTCCCCAAAACGC | Amplification of 458 bp to confirm rpfR deletion (2,641 bp in the wild type) |
| conf_rpfR_final_R | AGCCGAAGCTGAACGGGATTC | |
| Deletion of the bcsB gene in P0213-1 | ||
| bcsB_spacer_F | GTGGTGCAGGACAATTCGACGCTC | bcsB spacer for gene deletion in P0213-1 |
| bcsB _spacer_R | AAACGAGCGTCGAATTGTCCTGCA | |
| bcsB _P1 | TTTTGAGATCTGTCCATACCCATGGTCTAGATTGAACGTGTCGGTGATCTG | Amplification of 940 bp region upstream bcsB |
| bcsB _P2 | CACCATCCGTTACATCCCCGCAGCTGCATGGAATGTG | |
| bcsB _P3 | CACATTCCATGCAGCTGCGGGGATGTAACGGATGGTG | Amplification of 912 bp region downstream bcsB |
| bcsB _P4 | TCTGAATGGCGGGAGTATGAAAAGTCTCGAGATGCCGACCCACAGATAGAC | |
| conf_ bcsB_CR_F | GTTTTCGTTCGTGGAGGCAT | Amplification of 2,028 bp to confirm the mutant (4.275 bp in the wild type) |
| conf_ bcsB_CR_R | TCGTGTCGACTTTCTCCGG | |
| conf_bcsB _final_F | CGCCGTTTCGATAAATGAAT | Amplification of 223 bp to confirm bcsB deletion (2,470 bp in the wild type) |
| conf_bcsB _final_R | CCGCACCATCCGTTACAT | |
| Deletion of the bceF gene in P0213-1 and ATCC 17616 | ||
| sg_bceF_ATCC_F | GTGGTCCGTCGGAGTTGCTGATGT | bceF spacer for gene deletion in ATCC 17616 |
| sg_bceF_ATCC_R | AAACACATCAGCAACTCCGACGGA | |
| sg_bceF_F | GTGGCAACATCCATCTGATCGATT | bceF spacer for gene deletion in P0213-1 |
| sg_bceF_R | AAACAATCGATCAGATGGATGTTG | |
| bceF_P1 | TTTTGAGATCTGTCCATACCCATGGTCTAGAGATCACCCGGAGCTGACG | Amplification of 971 bp region upstream bceF |
| bceF_P2 | CATGCGCTCAGGTATTCGTGTTGCGTGTTCACCATTCGTT | |
| bceF_P3 | AACGAATGGTGAACACGCAACACGAATACCTGAGCGCATG | Amplification of 1,189 bp region downstream bceF |
| bceF_P4 | TCTGAATGGCGGGAGTATGAAAAGTCTCGAGCGTGGTGATAGCCGTACAGA | |
| conf_bceF_CR_F | GTGTATCACGTCGGCCCG | Amplification of 2,222 bp to confirm the mutant (4,412 bp in the wild type) |
| conf_bceF_CR_R | GAATTCGTCCGATTCCGCG | |
| conf_bceF_F | CTTCTACATGCGGCAGATCA | Amplification of 222 bp to confirm the mutant (2,412 bp in the wild type) |
| conf_bceF_R | GGTAGGTCGGAACGAGCAC | |
| Deletion of the cepR gene in P0213-1 and ATCC 17616 | ||
| sg_cepR_P0213-1_F | GTGGCCATGCCGATCGGCTCACAC | cepR spacer for gene deletion in P0213-1 |
| sg_cepR_P0213-1_R | AAACGTGTGAGCCGATCGGCATGG | |
| sg_cepR_F | GTGGACGGCTGGATGGCGCACTAT | cepR spacer for gene deletion in ATCC 17616 |
| sg_ cepR _R | AAACATAGTGCGCCATCCAGCCGT | |
| cepR _P1 | TTTTGAGATCTGTCCATACCCATGGTCTAGACACAACCGCACATCTCGC | Amplification of 939 bp region upstream cepR |
| cepR _P2 | AGGGCGTATCGATGAGTCCCCAGCGCAGTTCCATTCTTT | |
| cepR _P3 | AAAGAATGGAACTGCGCTGGGGACTCATCGATACGCCCT | Amplification of 936 bp region downstream cepR |
| cepR _P4 | TCTGAATGGCGGGAGTATGAAAAGTCTCGAGCGCCTACCGTTTCACACTG | |
| conf_ cepR _F | AACAGTCGTTCCATGCTCG | Amplification of 2,225 bp to confirm the mutant (2,909 bp in the wild type) |
| conf_ cepR _R | GTGGACAGTTGCAGCACTC | |
| conf_ cepR_final_F | TTTCTGACAGGCGCACATAG | Amplification of 270 bp to confirm the mutant (954 bp in the wild type) |
| conf_ cepR_final_R | GGAGCCGATGATGGAGTGA | |
| Insertion of gfp gene in ATCC 17616 | ||
| sg_IG_F | GTGGAAGGTTGCGGATCTGAAACA | IG region spacer for gfp gene insertion in ATCC 17616 |
| sg_IG_R | AAACTGTTTCAGATCCGCAACCTT | |
| IG_P1 | TTTTGAGATCTGTCCATACCCATGGTCTAGACTTTCTGAAGTCGCCGCG | Amplification of 709 bp region upstream of the Cas9 cutting site |
| IG_P2 | TGATTAATTGTCAACAGCTCCGATGGGCATGCGAAAGTC | |
| IG_P3 | GACTTTCGCATGCCCATCGGAGCTGTTGACAATTAATCA | Amplification of a 797 bp fragment containing the gfp coding sequence and promoter |
| IG_P4 | CGATGGGCATGCGAAAGTCCTATTTGTATAGTTCATCCA | |
| IG_P5 | TGGATGAACTATACAAATAGGACTTTCGCATGCCCATCG | Amplification of 770 bp region downstream of the Cas9 cutting site |
| IG_P6 | TCTGAATGGCGGGAGTATGAAAAGTCTCGAGCAGAAGTACAAGGCCGACAG | |
| Conf_gfp_F | GAATCGCTGAAGGGCAAGTC | Amplification of 2,276 bp to confirm gfp gene insertion (1,922 bp in the wild type) |
| Conf_gfp_R | CACGCTGTTTCTCGACGAA | |
| Conf_gfp_final_F | CCATCGGAGCTGTTGACAAT | Amplification of 936 bp to confirm gfp gene insertion (no amplification in the wild type) |
| Conf_gfp_final_R | GGACCATGTTTCAGATCCGC | |
a
Restriction sites are in italic and underlined.