| Outer primers: |
| Forward outer primer: (NdeI site underlined) |
5′-GCAGCCATATGGGAGGTTCTCCTTCCCTCATAGATGTTGTGGTTGTG-3′ |
| Reverse outer primer: (BamHI site underlined) |
5′-AGCCGGATCCTCGAGCTACTAACCTTCAATGCTGAAAATT TGTTC-3′ |
|
Inner primers: (mutation sites are underlined) |
| S214A-forward: |
5′-GCAACATCCCAGACAGGTCAATATGGTGGGG-3′ |
| S214A-reverse: |
5′-CCCCACCATATTGACCTGTCTGGGATGTTGC-3′ |
| Y216G-forward: |
5′-CCCAGACATCCCAAGGTGGTGGGGACCTCAC-3′ |
| Y216G-reverse: |
5′-GTGAGGTCCCCACCACCTTGGGATGTCTGGG-′ |
| G217K-forward: |
5′-CAGACATCCCAATATAAAGGGGACCTCACAAAC-3′ |
| G217K-reverse: |
5′-GTTTGTGAGGTCCCCTTTATATTGGGATGTCTG-3′ |
| G218L-forward: |
5′-GACATCCCAATATGGTCTGGACCTCACAAACAC-′ |
| G218L-reverse |
5′-GTGTTTGTGAGGTCCAGACCATATTGGGATGTC-3′ |
| D219A-forward: |
5′-CAATATGGTGGGGCACTCACAAACACATTCGGAGC-3′ |
| D219A-reverse: |
5′-GCTCCGAATGTGTTTGTGAGTGCCCCACCATATTG-3′ |
