Table 4 Oligonucleotide primers used for the site-directed mutagenesis of SK and AK1
From: The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase
Mutant | 5’-phosphorylated primer sets, indicating silently mutated restriction enzyme site (underlined) and new codon (bold) | Restriction enzyme site |
|---|---|---|
AK1-T23V | T23V-F: 5'-AGGGCGTC CAGTGTGAGAAGA-3' | Bam HI |
T23V-R: 5'-TCCCGGATCC AGGCCCA-3' | ||
AK1-T39V | T39V-F: 5'-CTACACCCACTTAAG CGTC GGGGAC-3' | Afl II |
T39V-R: 5'-CCATACTTCTGCACGATCTTCTCAC-3' | ||
AK1-R97A | R97A-F: 5'-ATTGATGGATATC CGGCT GAGGTGC-3' | Eco RV |
R97-R: 5'-CAGGAAGCCTTTGGAAGTATTGACTTTG-3' | ||
AK1-R97K | R97K-F: 5'-ATTGATGGATATC CGAAG GAGGTGCA-3' | Eco RV |
R97-R: 5'-CAGGAAGCCTTTGGAAGTATTGACTTTG-3' | ||
AK1-R97Q | R97Q-F: 5'-ATTGATGGATATC CGCAG GAGGTGC-3' | Eco RV |
R97-R: 5'-CAGGAAGCCTTTGGAAGTATTGACTTTG-3' | ||
AK1-R128A | R128A-F: 5'-ACCCAGGCT CTCTTGAAACGCGT AGAGAC-3' | Mlu I |
R128-R: 5'-CATGGTCTCAGGGCCTGCGT-3' | ||
AK1-R128K | R128K-F: 5'-ACCCAGAAG CTCTTGAAACGCGT AGAGAC-3' | Mlu I |
R128-R: 5'-CATGGTCTCAGGGCCTGCGT-3' | ||
AK1-R128Q | R128Q-F: 5'-ACCCAGCAG CTCTTGAAACGCGT AGAGAC-3' | Mlu I |
R128-R: 5'-CATGGTCTCAGGGCCTGCGT-3' | ||
AK1-R132A | R132A-F: 5'-ACCCAGCGGCTCCTTAAG GCT GGAGAGACC-3' | Afl II |
R128-R: 5'-CATGGTCTCAGGGCCTGCGT-3' | ||
AK1-R132K | R132K-F: 5'-ACCCAGCGGCTCCTTAAG AAA GGAGAGACC-3' | Afl II |
R128-R: 5'-CATGGTCTCAGGGCCTGCGT-3' | ||
AK1-R132Q | R132Q-F: 5'-ACCCAGCGGCTCCTTAAG CAA GGAGAGACC-3' | Afl II |
R128-R: 5'-CATGGTCTCAGGGCCTGCGT-3' | ||
SK-K15I | K15I-F: 5'-CTGCCGGATCC GGCATA TCCACCAT-3' | Bam HI |
K15-R: 5'-GCCGACGAGAACCGCTTTGGGTG-3' | ||
SK-K15R | K15R-F: 5'-CTGCCGGGATCC GGCAGG TCCACCA-3' | Bam HI |
K15-R: 5'-GCCGACGAGAACCGCTTTGGGTG-3' | ||
SK-T17I | T17I-F: 5'-CTGCCGGGATCC GGCAAGTCCATA ATCGGGCG-3' | Bam HI |
K15-R: 5'-GCCGACGAGAACCGCTTTGGGTG-3’ | ||
SK-T17R | T17R-F: 5'-CTGCCGGGATCC GGCAAGTCCAGA ATCGGGCG-3' | Bam HI |
K15-R: 5'-GCCGACGAGAACCGCTTTGGGTG-3' | ||
SK-R110A | R110A-F: 5'-GGCGTGCGCGCA ACCGGCGGC-3' | Eco RV |
R110-R: 5'-CTCGGCGGCGGATATC TCCAGGTAG-3' |