TABLE 1.
Mutant primers used for overlap extension PCR
| Sequencea | Description or mutations | Direction |
|---|---|---|
| GCGAGCCCTCAGATCCTGCATATAA | 5′ flanking primer for residues 40 through 205 | |
| CGTGGCCCATCCCAGAAAGACGGAA | 3′ flanking primer for residues 40 through 205 | |
| CCGCCAGcTTCTGcCATGGcTCTGAA | D40A-D42A-D44A | Forward |
| TTCAGAgCCATGgCAGAAgCTGGCGG | D40A-D42A-D44A | Reverse |
| TGACGGcATGGCGCCGTGTGAGTgcGGCCC | E66A-K72A | Forward |
| GGGCCgcACTCACACGGCGCCATgCCGTCA | E66A-K72A | Reverse |
| GTGCAATTTGcGgcGGGAGcGAGCTACTTC | E83A-K84A-E86A | Forward |
| GAAGTAGCTCgCTCCCgcCgCAAATTGCAC | E83A-K84A-E86A | Reverse |
| AGTCAGATTCGCgcAgcACTGATTCAGAGA | E114A-K115A | Forward |
| TCTCTGAATCAGTgcTgcGCGAATCTGACT | E114A-K115A | Reverse |
| GGGAACgcGGTGGTGGcTGcGTGCTACATC | K146A-D149A-E150A | Forward |
| GATGTAGCACgCAgCCACCACCgcGTTCCC | K146A-D149A-E150A | Reverse |
| AATCTCACGGcGCGTgcACGGTTGGTGGCG | E184A-K186A | Forward |
| CGCCACCAACCGTgcACGCgCCGTGAGATT | E184A-K186A | Reverse |
| ACGCAGGCGCAGAACGCAGCGAATCAGAAT | E201A-K204A-E205A | Forward |
| ATTCTGATTCGCTGCGTTCTGCGCCTGCGT | E201A-K204A-E205A | Reverse |
| TATGGAACAGTATTTAAGCGCCTGT | 5′ flanking primer for residues 378 through 483 | |
| GAGAGAGTGTCCTCGAGCCAATCT | 3′ flanking primer for residues 378 through 483 | |
| CTGGTGGGcGGcGGGGgcGATGACCG | E378A-E379A-K381A | Forward |
| GGCGGTCATCgcCCCCgCCgCCCAC | E378A-E379A-K381A | Reverse |
| GCGCGTGGcCCAGgcATGCgcGTCCTCGGC | D402A-K404A-K406A | Forward |
| GCCGAGGACgcGCATgcCTGGgCCA | D402A-K404A-K406A | Reverse |
| GCCGTTGCAAGcCCGGATGTTCgcATTTG | D443A-K447A | Forward |
| CAAATgcGAACATCCGGgCTTGCAACGGC | D443A-K447A | Reverse |
| CTCACCCGCCGTCTGGcTCATGcCTTTG | D455A-D457A | Forward |
| CCCAAAGgCATGAgCCAGACGGCGGGT | D455A-D457A | Reverse |
| ACCgcGCAGGcAGTCgcAGcCTTTTTCCG | K463A-E465A-K467A-D468A | Forward |
| CGGAAAAAGgCTgcGACTgCCTGCgcGGT | K463A-E465A-K467A-D468A | Reverse |
| GACTTTTTCCGGTGGGCAgcGGcTCACG | K474A-D475A | Forward |
| CGTGAgCCgcTGCCCACCGGAAAAAGTC | K474A-D475A | Reverse |
| GTGGTTGcGGTGGcGCATGcATTCTACG | E479A-E481A-E483A | Forward |
| GACGTAGAATgCATGCgCCACCgCAACCAC | E479A-E481A-E483A | Reverse |
a
Bold lowercase letters indicate the mutation sites. Forward oligonucleotides and their reverse complements are written 5′ to 3′. Lysine (AAA and AAG), aspartic acid (GAC and GAT), and glutamic acid (GAA and GAG) codons converted to alanine codons (GCA, GCT, GCC, and GCG) are underlined in the forward strands.