Table 1.
Plasmids and primers for overexpression.
| Plasmid | Forward primer | Reverse primer | Template |
|---|---|---|---|
| pIMPI-T50Na | CATATACAGAATAAAAATAACTGTCCC | GGGACAGTTATTTTTATTCTGTATATG | pIMPI-WT |
| pIMPI-T50Q | CATATACAGAATAAACAAAACTGTCCC | GGGACAGTTTTGTTTATTCTGTATATG | pIMPI-WT |
| pIMPI-T50R | CAGAATAAACGAAACTGTCCCATC | GATGGGACAGTTTCGTTTATTCTG | pIMPI-T50Q |
| pIMPI-T50Y | CATATACAGAATAAATATAACTGTCCC | GGGACAGTTATATTTATTCTGTATATG | pIMPI-T50N |
| pIMPI-I54M | CTGTCCCATGATTAATATAAGATGTAATGACAAGTGC | GCACTTGTCATTACATCTTATATTAATCATGGGACAG | pIMPI-WT |
| pIMPI-I55R | GTCCCATCCGTAATATAAGATGTAATG | CATTACATCTTATATTACGGATGGGAC | pIMPI-I55W |
| pIMPI-I55W | CAAACTGTCCCATCTGGAATATAAGATGTAATGAC | GTCATTACATCTTATATTCCAGATGGGACAGTTTG | pIMPI-WT |
| pIMPI-I55Y | CAAACTGTCCCATCTATAATATAAGATGTAATG | CATTACATCTTATATTATAGATGGGACAGTTTG | pIMPI-WT |
| pIMPI-I57F | CTGTCCCATCATTAATTTTAGATGTAATGACAAGTGC | GCACTTGTCATTACATCTAAAATTAATGATGGGACAG | pIMPI-WT |
| pIMPI-I57Y | CTGTCCCATCATTAATTATAGATGTAATGACAAGTGC | GCACTTGTCATTACATCTATAATTAATGATGGGACAG | pIMPI-WT |
| pIMPI-R58E | CATTAATATAGAATGTAATGACAAGTGC | GCACTTGTCATTACATTCTATATTAATG | pIMPI-WT |
| pIMPI-T50Y + I55R | CATATACAGAATAAATATAACTGTCCC | GGGACAGTTATATTTATTCTGTATATG | pIMPI-I55R |
| pIMPI-T50Y + I55R + I57F | CCCATCCGTAATTTTAGATGTAATGACAAGTGC | GCACTTGTCATTACATCTAAAATTACGGATGGG | pIMPI-T50Y + I55R |
a
This mutant was used as an intermediate to prepare T50Y and was not tested for activity. Only single nucleotides were exchanged in each reaction. For the double and triple mutants, a corresponding ancestral plasmid was used as the template.