TABLE 1.
Oligonucleotides
| Oligonucleotide | Description |
|---|---|
| HL38 | 5′-GGAAGAGGAATCCTGGC-3′: 5′-flanking primer, upstream of XhoI site, used to generate ΔA, ΔB, and ΔC mutations |
| HL39 | 5′-CCAATTGCTTCCAGTCTTTG-3′: 3′-flanking primer, downstream of AvrII site, used to generate ΔA, ΔB, and ΔC mutations |
| HL87 | 5′-CGATAATTGAACGCTACACC-3′: 5′-flanking primer, upstream of AvrII site, used to generate ΔD mutation |
| HL88 | 5′-TTGTTTATTTGACATGTATGGC-3′: 3′-flanking primer, downstream of BsrGI site, used to generate ΔD mutation |
| HL205 | 5′-CAAGTCGTTCTATAGCGTAGCCGGCATCGGAATGTTCACCAAAAAA-3′: bottom-strand primer used to create ΔB deletiona |
| HL206 | 5′-TTTTTTGGTGAACATTCCGATGCCGGCTACGCTATAGAACGACTTG-3′: top-strand primer used to create ΔB deletion |
| HL207 | 5′-CAATATCCACCATTTCCATCGCCGGCCATTCTTGGAATAGCGAGT-3′: bottom-strand primer used to create ΔA deletion |
| HL208 | 5′-ACTCGCTATTCCAAGAATGGCCGGCGATGGAAATGGTGGATATTG-3′: top-strand primer used to create ΔA deletion |
| HL209 | 5′-AAAGTTAAAAATCTTGTCTATATTGCCGGCCTGTTTAATCAGATAGGGTG-3′: bottom-strand primer used to create ΔC deletion |
| HL210 | 5′-CACCCTATCTGATTAAACAGGCCGGCAATATAGACAAGATTTTTAACTTT-3′: top-strand primer used to create ΔC deletion |
| HL211 | 5′-TGAAACATTTGTTTTCTTTGGGCCGGCAAACTTATTGTTTTTCCAATTG-3′: bottom-strand primer used to create ΔD deletion |
| HL212 | 5′-CAATTGGAAAAACAATAAGTTTGCCGGCCCAAAGAAAACAAATGTTTCA-3′: top-strand primer used to create ΔD deletion |
a
Top- and bottom-strand primers were used to introduce an NgoMI site, as well as to delete amino acids. Deletion constructs were created by fusion PCR, including amplification with HL38 (for bottom strands corresponding to HL205, HL207, and HL209), HL39 (for top strands corresponding to HL206, HL208, and HL210), HL87 (for a bottom strand corresponding to HL211), and HL88 (for a top strand corresponding to HL212).