TABLE 2.
Sequences of primers used to prepare IMPDH ΔCBS mutants
A set of three primers was utilized for each construct, where F, Del R, and R designate forward, deletion reverse, and reverse primer, respectively. Inserted connecting sequence (resulting in G, GG, or SGG amino acid sequence) in deletion reverse primer is shown in bold type. NA, not applicable.
| Construct | Primer direction | Deleted residue(s) | Inserted residue(s) | PCR primer sequence 5′to 3′ |
|---|---|---|---|---|
| BaIMPDHΔS | F | NA | NA | TACTTCCAATCCAATGCCATGTGGGAATCTAAATTTGTTAAAGAAGGTCT |
| BaIMPDHΔS | Del R | Val-95–Thr-200 | Gly | GAATTTGGGAATTCAATTACTTTTTCAATATCTTTTATACCGCCACTTTCAGAACGTTTTACTTTATCAA |
| BaIMPDHΔS | R | NA | NA | TTATCCACTTCCAATGTTATTATAATGAGTAGTTTGGAGCCTCTTTTGTAATTT |
| BaIMPDHΔL | F | NA | NA | TACTTCCAATCCAATGCCATGTGGGAATCTAAATTTGTTAAAGAAGGTCT |
| BaIMPDHΔL | Del R | Glu-92–Arg-220 | GG | ACACCAACTGCTGCTCCAACTAATAAACCACCAGAACGTTTTACTTTATCAACTTGCTCGG |
| BaIMPDHΔL | R | NA | NA | TTATCCACTTCCAATGTTATTATAATGAGTAGTTTGGAGCCTCTTTTGTAATTT |
| CjIMPDHΔS | F | NA | NA | TACTTCCAATCCAATGCCATGAAAATTGTAAAAAGAGCTTTAACTTTTGAAGATGTAT |
| CjIMPDHΔS | Del R | Val-92–Thr-195 | Gly | ATCAGGATATTCTTTGCGTTTTTTAAGATCTTTTATACCCCCGCTTTCACTTTTTTTCACTCTTTTTA |
| CjIMPDHΔS | R | NA | NA | TTATCCACTTCCAATGTTATCATTGATGATTGACCTTGTAATTTGGTG |
| ClpIMPDHΔL | F | NA | NA | TACTTCCAATCCAATGCCATGGCAAGAATATTAAAAACAGCATATACATTTGAT |
| ClpIMPDHΔL | Del R | Gln-89–Arg-215 | SGG | CACCGATTGAAGCTCCACATAAAAGACCACCACTTCTTTTTACTCTATCAACTTCTCTTGCTTGAT |
| ClpIMPDHΔL | R | NA | NA | TTATCCACTTCCAATGTTATTATTGGTTAACACTGTAGTTTGGTGCTT |
| VcIMPDHΔL | F | NA | NA | TACTTCCAATCCAATGCCTTGCACATGCTACGAATCGCAAAAGAA |
| VcIMPDHΔL | Del R | Phe-91–Arg-219 | SGG | AGCGGCACCAACACGCAGACCACCACTGATTTTCACTTGGTGAACTTGAGCC |
| VcIMPDHΔL | R | NA | NA | TTATCCACTTCCAATGTTATTAACCCAGACGGTAGTTTGGTGC |