Table 2 Primers used in the present study.
Primer | Sequence 5′-3′ | Amplified DNA |
|---|---|---|
Phusion PCR for carB disruption | ||
carBcrispfuz1 | CTGTGCAGGTGCCAGCGCATCACC | carB promoter and coding sequence upstream from protospacer |
carBcrispfuz2 | GGTGGTCTTGAATGCGCTCGTCCAG | |
carBcrispfuz3 | TGGAGCTGCTGCGATGCGACAAC | carB coding sequence downstream from protospacer |
carBcrispfuz4 | GTCTTGCTCTTCATATGACCAATAG | |
carBcrispfuz5 | CCGATCTGGACGAGCGCATTCAAGACCACCTGCCTCAGCATTGGTACTTG | pyrG with own promoter and terminator sequences |
carBcrispfuz6 | GTAGTTGTTGTCGCATCGCAGCAGCTCCAGTACACTGGCCATGCTATCG | |
carBcrispfuz7 | CTTGTGGTAGACAATGTAGTTGTC | phusion pcr product |
carBcrispfuz8 | CGTCAAAGCTCTCCTGATAAGCCTC | |
Primers for analysis of the carB mutants | ||
MccarRPfw | ATGCTGCTCACCTACATGGAAG | carRP |
MccarRPrev2 | TTAAATGGTATTTAGATTTCTCA | |
MccarB1 | ATGTCCAAGAAACACATTGTCATTA | carB |
MccarB2 | TTAAATGACATTAGAGTTATGAACG | |
MccarBupfw | TAGCCAATGACAGCGGTGACGC | upstream region from protospacer |
MccarBuprev | GTTGTCGCATCGCAGCAGCTCCA | |
MccarBdwfw | TGGAGCTGCTGCGATGCGACAAC | downstream region from protospacer |
MccarBdwrev | CGTCAAAGCTCTCCTGATAAGCCTC | |
Phusion PCR for hmgR 2 disruption | ||
h2p1 | ACGGAGCAAGTTTACATTCATACC | hmgR2 promoter and coding region upstream from protospacer |
h2p2 | GATTGGCGGTCTATCATATTTCAG | |
h2p3 | CTCAACATCTCTTGTACTATGCCC | hmgR2 coding sequence downstream from protospacer |
h2p4 | ATACGTTTGTACCGATGAAGGGA | |
h2pyrG5 | CTCATTGCTGAAATATGATAGACCGCCAATCGTACAATTCCCTGCCTTCTGGAAG | pyrG with own promoter and terminator seqences |
h2pyrG6 | CGATACAGGGCATAGTACAAGAGATGTTGAGCGCGTGAAAGACCTGCCTTGACTC | |
h2p7 | TGTGAAATTGGCCTATAATCAGCCT | phusion pcr product |
h2p8 | GGACAGACATTCCATCGTATACAG | |
Primers for analysis of the hmgR 2 mutant | ||
h2cdns1 | ATGTTGAAAAACGTCAAAAAAGAT | hmgR2 |
h2cdns2 | CTATGATTTAATACAACTTCCA | |
Primers used in the qPCR experiment | ||
MCactinF | CACTCCTTCACTACCACCGCTGA | 117 bp of actin |
MCactinR | GAGAGCAGAGGATTGAGCAGCAG | |
H2_RT_F | CTCGTATCATCTGTGCCTCTG | 107 bp of hmgR2 |
H2_RT_R | AGCAGTGTTACGGTTGTGAG | |
