TABLE 1.
Primers used in this study
| Category | Primer | Sequencea | Cut site(s) |
|---|---|---|---|
| PBin-mtx1 fusion | BinmutF | GGAGATGAAGAAACCATGGGCGATTCAAAAGAC | NcoI |
| BinmutR | GTCTTTTGAATCGCCCATGGTTTCTTCATCTCC | NcoI | |
| mtx1F | CCATGGCTTCACCTAATTCTCCAAAAG | NcoI | |
| mtx1R | CCATGGTCATTACTATCTAGGTTCTACAC | NcoI | |
| mtx1 mutants | MtxF | CCGGAATTCCCGGATCCTCACCTAATTCTCCAAAAGAT | EcoRI, BamHI |
| MtxR | GGCCCGGGATCCTATCTAGGTTCTACACCTAATG | SmaI, BamHI | |
| MutLeuF | GGATTCTAAAGGTTTGATACTAGATTTAG | ||
| MutGlyF | GGATTCTAAAGGTGGTATACTAGATTTAG | ||
| MutArgF | GGATTCTAAAGGTCGTATACTAGATTTAG | ||
| MutKKF | GGAAATAATATGGATAAGAAAGGTAAGATACTAGATTTAG | ||
| MutLeuR | CTAAATCTAGTATCAAACCTTTAGAATCC | ||
| MutGlyR | CTAAATCTAGTATACCACCTTTAGAATCC | ||
| MutArgR | CTAAATCTAGTATACGACCTTTAGAATCC | ||
| MutKKR | CTAAATCTAGTATCTTACCTTTCTTATCCATATTATTTCC | ||
| Mtx2 production | mtx2N15TF | GAAGGATCCATGGAATTGTTTGTAAACGGAAGTATATATACG | BamHI, NcoI |
| mtx2N15TR | GGGCTCGAGTTATTTAAAAGAAATTTCTTTAACATCTATTA | XhoI | |
| Domain II variants | A | GGATCCTTTTGATGGTAGTTTTCGTGGA | BamHI |
| B | GACGTCAGTATAAATGGTTATACTATTAAG | AatII | |
| C | AATTATTGGTCAGGGCATCAAA | ||
| D | ATATAAAGGTGAAGATAATGTTCT | ||
| E | CTGTTTGTCCTTGATGGAACG | ||
| F | CAGCATTGTAACATGACTCAATC | ||
| G | TTGAGAGCTCCAACGTTTTCTTGGCAGCATCGCAGTGCGGCCG | SacI, NotI | |
| H | CGGCCGCACTGCGATGCTGCCAAGAAAACGTTGGAGCTCTCAA | NotI, SacI | |
| 1.1 | CTTAATAGTATAACCATTTATACTGACGTC(NNS)4AATTTATTGGTCAGGGCATCAAA | AatII | |
| 1.2 | CTTAATAGTATAACCATTTATACTGACGTC(NNS)3AATTTATTGGTCAGGGCATCAAA | AatII | |
| 1.3 | CTTAATAGTATAACCATTTATACTGACGTC(NNS)5AATTTATTGGTCAGGGCATCAAA | AatII | |
| 2.1 | AGAACATTATCTTCACCTTTATAT(NNS)13CTGTTTGTCCTTGATGGAACG | ||
| 2.2 | AGAACATTATCTTCACCTTTATAT(NNS)12CTGTTTGTCCTTGATGGAACG | ||
| 2.3 | AGAACATTATCTTCACCTTTATAT(NNS)14CTGTTTGTCCTTGATGGAACG | ||
| 3.1 | GATTGAGTCATGTTACAATGCTG(NNS)9TTGAGAGCTCCAACGTTTTCT | ||
| 3.2 | GATTGAGTCATGTTACAATGCTG(NNS)8TTGAGAGCTCCAACGTTTTCT | ||
| 3.3 | GATTGAGTCATGTTACAATGCTG(NNS)10TTGAGAGCTCCAACGTTTTCT | ||
| cry1Aa construction | N-termF | GGATCCTAAGTAGATTGTTAACACCCTG | BamHI |
| C-term F | GACGTCCATAGAGGCTTTAATTATTGGTC | AatII | |
| C-termR | GGATCCCTATTCCTCCATAAGGAGTAATTC | BamHI |
a
The sequences of the restriction enzymes noted in the fourth column are underlined.