TABLE 2.
Primer and function(s) | Sequence (5′→3′) | Primer | Sequence (5′→3′) |
---|---|---|---|
Chromosomal disruption, inactivation of tfdFI and tfdFII genes,a and overlapping RT-PCR | |||
tcpR1 | TCAGCTTCTTCCTGCTCGAT | tcpR2 | GGATACCAGCACATGCTTCTC |
tcpX1 | TCACGTGCTCGGCTGTTTGT | tcpX2 | ACCATGGCGGACTTGCTGTA |
tcpA1 | CCAAGAAGCACGACCTGA | tcpA2 | CTTGTTGGTGCCGATATG |
tcpC1 | AGGAGTTCATCCTGCTCAG | tcpC2 | CGCACGCCGAACACGGCATC |
tcpY1 | CGTGGTACCGATCGTGTCT | tcpY2 | GATATCGACCTTGGGAGTCG |
tcpD1 | GGCTCGGAGATGACTACGAT | tcpD2 | CATGGGCGTACAAACCTTCT |
ReutB4129 1 | ATCTACGATCCCGCACTGAC | ReutB4129 2 | ATGGCACAGCTTGTGATGAA |
ReutB4694 1 | GGTCTACGATCCCGAACTGA | ReutB4694 2 | GTATGGCACAGCTTGTGGTG |
ReutC5982 1 | ACCTGCACCAACAGCAGAC | ReutC5982 2 | TCTAACACGGCGAAAAATCG |
FAD1 | TCGATGGTGTACATTGCGGG | FAD2 | AAGTCATCCGCACTGGCTTC |
MUTF1FW | GACCCTTCATGAAGAAGTTCACGCTTGACTACCTGAGCCCGTGTAGGCTGGAGCTGCTTC | MUTF1RE | GCGGAGTTGCAGGTCACATTATTTGAAATCCGGTCTTCGCCATTCCGGGGATCCGTCGACC |
MUTF2FW | CCGGCGATCTGAATGAATTCGTTGCGCACTTCTGGCCGGTGTGTAGGCTGGAGCTGCTTC | MUTF2RE | AGAGGTCCATGGGATGTCCGGTTCACGCCGGCATTTCTCCATTCCGGGGATCCGTCGACC |
FORF1 | ATGAAGAAGTTCACGCTTGACTACCTGAGC | REVF1 | ACCGTACTAAACGCGGAGTG |
FORF2 | GCACTAGTAGTGACCGGCGAT | REVF2 | CTTATCGATAGGTCGGGTCG |
TcpA3 | GTGCAGGTCGTAGAAGTCG | tcpAend | GAGGGCCACGACAGCGAATAC |
TCPout | AACCTTCCACATTTTGTGCC | ||
RT-PCR MARs | |||
RTtfdFI1 | ACGCGAGTTAGCGAAGGATA | RTtfdFI2 | GAGATAGCAAGCGGCAAATC |
RTtfdFII1 | GAATTCGTTGCGCACTTCTG | RTtfdFII2 | GGCAAGGAGGTCAGGTGAT |
RTtcpD1 | TCGCGCACGCAGCAGAAGGTTTGT | RTtcpD2 | ACGCGGGTTCGGGTACTGGTTCTG |
RT ReutB4129 1 | ATCTACGATCCCGCACTGAC | RT ReutB4129 2 | ATGGCACAGCTTGTGATGAA |
RT ReutB4694 1 | GGTCTACGATCCCGAACTGA | RT ReutB4694 2 | GTATGGCACAGCTTGTGGTG |
RT ReutC5982 1 | ACCTGCACCAACAGCAGAC | RT ReutC5982 2 | TCTAACACGGCGAAAAATCG |
RT358F | CCTACGGGAGGCAGCAG | RT907R | CCGTCAATTCTTTTRAGTTT |
Transcription start mapping (RACE) | |||
X1 | CGCTCACGGAACAAACAGC | X2 | GTGGCGATGACGGTCACG |
X3 | ACTGCACGCGACAAGGCG | AUAP | GGCCACGCGTCGACTAGTAC |
AAP | GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG | ||
Constitutive expression of tcp gene sequencesb | |||
tcpRlacZ | ATCGAAGGTCAGCAATACGG | tcpB-XbaI | ATGCTGTCTAGACTCTTGCATCACTGGACTCC |
tcpR-EcoRI | GCTAATGAATTCGCATCATGGACACTATTCCC | tcpR-HindIII | CCGCGGAAGCTTATCGAAGGTCAGCAATACGG |
tcpX-EcoRI | GGAGGAGAATTCCATGTCGTCCGCAGTCTTC | tcpX-XbaI | CGCGGGTCTAGACGCTTCAAGTCGCGCTAGG |
tcpA-EcoRI | AGGAGAGAATTCGATGATTCGCACTGGCAAGC | tcpA-XbaI | CTGCGTTCTAGACGGAAGATCTTGTCAAGCAG |
tcpC-EcoRI | CGGGAGGAATTCGATGCAAGAGTATGACCAGC | tcpC-HindIII | GACCTTAAGCTTATCTGTCGAACCCATTTGCC |
tcpD-EcoRI | TTTGACGAATTCCCCTACGATGAAAGCATTCC | tcpD-HindIII | GTATTGAAGCTTAACCTTCCACATTTTGTGCC |
Primers to inactivate either tfdFI or tfdFII genes (MUTF1FW, MUTF1RE, MUTF2FW, and MUTF2RE) contain nearly 40-bp homology extensions of the tfdFI or tfdFII gene sequence (in boldface) and nearly 20-bp priming sequences for pKD4 (6).
Primers include a restriction site which is underlined.