TABLE 1.
Site-directed mutagenesis primers
| Residue | Primera | Restriction siteb |
|---|---|---|
| C49 | 152-CATACTGGAATAGGTAACCAAGGCAGC-178 | BstEII |
| C76 | 236-AATTCATCTCTTGGGCCCATCCGTGGG-262 | ApaI |
| C109 | 331-GCCTTTTATTTCAGGATCCCACTTGGAATGC-361 | BamHI |
| C146 | 443-GCCTTAATGAGCGGGCCCGTCGGTGAAGCT-472 | ApaI |
| C169 | 507-CTTGGTCAGCTAGCGCAGGACATGGATGGAG-536 | NheI |
| C218 | 656-GAGTCTGAATGCACCGGAGTAAATGGTTC-684 | BsmI |
| C264 | 797-CACTACGAGGAAGGATCCTGTTACCC-822 | BamHI |
| C266 | 805-GGAATGTTCCGGATATCCGGATACCGGCAAAG-836 | BspEI |
| C303 | 917-GGATACATCGGATCCGGGGTTTTCG-941 | BamHI |
| C320 | 965-GGAACAGGCAGCGGGGGCCCAGTGTCT-991 | ApaI |
| C406 | 1222-CTGTATGAGGCCCGGGTTCTGGGTTG-1247 | SmaI |
a
The indicated deoxyoligonucleotide and a complementary deoxyoligonucleotide were used for mutagenesis reactions. Boldface characters indicate differences between the primer and the template, pGEM-NAm1; underlines indicate codons mutated from cysteine to glycine.
b
To facilitate the identification of mutant clones, the restriction sites were introduced without changing the amino acid sequence.