TABLE 2.
PCR primers used in this work
| 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 |
a
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).
b
Primers include a restriction site which is underlined.