TABLE 1.
Primers and mutant constructs used in this study
| Constructs | Primers | Templates | Expression system |
|---|---|---|---|
| Single pYES2-ADH-3A4-CYC mutants | |||
| T264A | Sense, GTCGCCTCGAAGATgCACAAAAGCACCGAG | pYES2-ADH-3A4-CYC | Yeast |
| Antisense, CTCGGTGCTTTTGTGcATCTTCGAGGCGAC | |||
| S420A | Sense, GTTCCTCCCTGAAAGATTCgcCAAGAAGAACAAGGACAAC | pYES2-ADH-3A4-CYC | Yeast |
| Antisense, GTTGTCCTTGTTCTTCTTGgcGAATCTTTCAGGGAGGAAC | |||
| S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pYES2-ADH-3A4-CYC | Yeast |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| Double pYES2-ADH-3A4-CYC mutants | |||
| T264A/S420A | Sense, GTTCCTCCCTGAAAGATTCgcCAAGAAGAACAAGGACAAC | pYES2-ADH-3A4T264A-CYC | Yeast |
| Antisense, GTTGTCCTTGTTCTTCTTGgcGAATCTTTCAGGGAGGAAC | |||
| T264A/S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pYES2-ADH-3A4T264A-CYC | Yeast |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| S420A/S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pYES2-ADH-3A4TS420A-CYC | Yeast |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| Triple pYES2-ADH-3A4-CYC mutants | |||
| T264A/S420A/S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pYES2-ADH-3A4T264AS420A-CYC | Yeast |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| Single, double, or triple pCW3A4His6 mutants | |||
| T264A | Sense, GTCGCCTCGAAGATgCACAAAAGCACCGAG | pGEM-3A4-His6 | DH5αF |
| Antisense, CTCGGTGCTTTTGTGcATCTTCGAGGCGAC | |||
| S420A | Sense, GTTCCTCCCTGAAAGATTCgcCAAGAAGAACAAGGACAAC | pGEM-3A4-His6 | DH5αF |
| Antisense, GTTGTCCTTGTTCTTCTTGgcGAATCTTTCAGGGAGGAAC | |||
| S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pGEM-3A4-His6 | DH5αF |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| T264A/S420A | Sense, GTTCCTCCCTGAAAGATTCgcCAAGAAGAACAAGGACAAC | pGEM-3A4-T264A-His6 | DH5αF |
| Antisense, GTTGTCCTTGTTCTTCTTGgcGAATCTTTCAGGGAGGAAC | |||
| T264A/S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pGEM-3A4-T264A-His6 | DH5αF |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| S420A/S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pGEM-3A4-S420A-His6 | DH5αF |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT | |||
| T264A/S420A/S478A | Sense, AGATCCCCCTGAAATTAgcCTTAGGAGGACTTCTTCAAC | pGEM-3A4-T264AS420A-His6 | DH5αF |
| Antisense, GTTGAAGAAGTCCTCCTAAGgcTAATTTCAGGGGGATCT |