Table 4.
Primers for construction of the arp mutagenic cassette and verification of allelic exchange
| Primer | Sequence (5′ > 3′) | Application |
|---|---|---|
| ARP01 |
GCCTTTCGTTAAGGTTTTGTTT |
amplify arp upstream homology |
| ARP02 |
GGAAATCTTCCTTGAAGCTCGGGTACAA |
SOEing arp upstream homology |
| |
GTTGTTCCTCCTAAATTAAATAAAAATAA |
to aadA cassette |
| ARP03 |
TACCCGAGCTTCAAGGAAG |
amplify aadA cassette |
| ARP04 |
GGTATATGTAATTTCGACTTTAAGTTAAAAAT |
SOEing arp downstream |
| |
CCGATTGTTTCATTTGCCGACTACCTTGGT |
homology to aadA cassett |
| ARP05 |
GAACAATCGGATTTTTTAACTTAAAGTCG |
amplify arp dowsteam homology |
| ARP06 |
ACCCCAGTAACTCAATTTCTAATTG |
amplify arp dowsteam homology |
| ARP07 |
TTTCTTGATTAGGGTAAAAAATTCT |
check integration at 5′ end |
| ARP08 |
GTCTTGTATTGTTGAACAAAACACTT |
check integration at 3′ end |
| ARP09 |
GTTTCCATATGAGGGAAGCG |
check integration within aadA |
| ARP10 | CCAAGCGATCTTCTTCTTGTC | check integration within aadA |