Table 2. Characteristics of primers used for single-site mutation of CAII.
List of the sense primers for single-site mutation CAII. Nucleotides that differ from the wild-type sequence are labelled bold. The antisense primers had the inverse complement sequence of the sense primers. Multiple mutations of CAII were created from single-site mutants using the primers listed in the table. CAII-H64A, which was also used in this study, was kindly provided by Dr. Robert McKenna, University of Florida, Gainesville, U.S.A.
| Mutation | Seq 5’→ 3’ | Tm (°C) |
|---|---|---|
| CAII-H3A | CCGAGGATGTCCGCTCACTGGGGGTACGGC | 76.3 |
| CAII-H4A | CCGAGGATGTCCCATGCCTGGGGGTACGGC | 76.3 |
| CAII-H3A/H4A | CCGAGGATGTCCGCTGCCTGGGGGTACGGC | 77.7 |
| CAII-K9A | GGGTACGGCGCACACAACGGACCTGAG | 72.6 |
| CAII-H10A | GGGTACGGCAAAGCCAACGGACCTGAG | 71.0 |
| CAII-H15A | GGACCTGAGGCCTGGCATAAGGACTTCC | 71.0 |
| CAII-H64K | CAACAATGGTAAAGCTTTCAACG | 57.1 |
| CAII-E69Q | CATGCTTTCAACGTGCAGTTTGAT | 59.3 |
| CAII-D71N | GGAGTTTAATGACTCTCAGG | 55.2 |
| CAII-D72N | TTTGATAACTCTCAGGACAAAGCA | 57.6 |