Table 1.
Forward oligonucleotides used in this study for the mutagenesis of the sor gene; the corresponding reverse-complimentary oligonucleotides required for the Quikchange method (Stratagene) are not shown.
| Oligonucleotide name | Oligonucleotide sequence* | Comment* |
|---|---|---|
| DelK fwd | CCGACTTCACTGCAGTTGTAggaggtggaATTC | Replacement of aa 139-148 with two |
| CAGTTATTTCACAACC | Gly residues; Addition of EcoRI site | |
| DelL fwd | CAAACATGCCTATAAACACTGGGggtggaATTT | Replacement of aa 129-151 with two additional |
| CACAACCATATGGAAA | Gly residues; Removal of PstI site | |
| R99A fwd | GGAGTTACTTATTCgcgCTATGCTATTCATGCGC | Addition of Bsh1236I site |
| R99IL fwd | AACTGGAGcTACTTATTCmtcCTATGCTATTCATG | Addition of AluI site |
| S226A fwd | AACCCTGGAgCACTTGAGCCcGATCCAAAT | Removal of PsuI site |
| S226T fwd | AACCCTGGAaCACTTGAGCCcGATCCAAAT | Removal of PsuI site |
| S226IL fwd | AACCCTGGamtACTTGAGCCcGATCCAAAT | Removal of PsuI site |
| MM296/297 fwd** | ATTAAATCCAryGryGGAAGGCACcTTCTGGAG- | Addition of BanI site |
| F133A fwd | TGAAATGACCGACgcaAtTGCGGTTGTA | Addition of MunI site, double mutant; T134I |
| F141A fwd | GTAGGAAAGAAGgcaGCAGAAGGAAAGCCT | Removal of PstI site |
| H166A fwd | GCCTTTGCAGAGgcgTCAGTAATTCC | Removal of Bsp1286I site |
| H277A fwd | TAAGACAAGTAgctGACGAAGTTTT | Removal of TatI site |
*Underlined, restriction sites specified in the comments column.
**Multiple mutants: M297 A, M296V, MM296/7VT, and MM296/7TT.