TABLE 1.
Oligonucleotide primers used in constructing expression plasmids and mutants
| Constructa | Primer sequence (5′-3′)b |
|---|---|
| spy0125 in pET28a | ttacatacatatgAAGACTGTTTTTGGTTTAGTAG |
| aagttctcgagTTAAACTCCTGTAGGAACAAC | |
| spy0125-N in pET28a-D | ctaggatccAAGACTGTTTTTGGTTTAGTAG |
| atgttctcgagTTAACCACCACTCAAAAGATT | |
| spy0125-C in pET28a-D | ctaggatccAATCAACCTCAAACGACTTCAG |
| atgttctcgagTTATGTAGGAACAACAGGCTC | |
| spy0128 in pET28a | ttacatacatatgGAGACTGTTGTAAACGGAGC |
| aagttctcgagTTATACTCCTGTTGGCACTTC | |
| spy0130 in pET28a | ttacatacatatgGAGAACCTCACTGCAAGCATT |
| aagttctcgagTTAACCAGTTGATGGCAAAATACC | |
| Δspy0125-N in pG+Host 9 | atgttctcgagTTATGTAGGAACAACAGGCTC |
| ttagtcgacACCAAAAACAGTCTTAGCAC | |
| attagtcgacAATCAACCTCAAACGACTT | |
| atgttctcgagAATCTGTTTTCCATCAATAA | |
| Δspy0125-C in pG+Host 9 | ttacatactgcagGTTatgtataatggacatccacaa |
| attagtcgacTGGAGGCATTGGTGGGTC | |
| attagtcgaccagctaactgaccttgatttc | |
| aagttctcgagATAGCCTTCAGAATCTGTTTC | |
| spy0128 in pG+Host 9 | attgaattccgatatggctcctagtgtaa |
| aagttctcgagGATGTGCATAGATTAATGAG | |
| spy0128K161A site-directed mutagenesis | TATAAAGAAGGTAGTgcGGTGCCAATTCAGTTC |
| GAACTGAATTGGCACCgcACTACCTTCTTTATA |
a
The expression plasmid or mutant constructed using the primer shown.
b
Lowercase letters indicate bases added to facilitate cloning or that were changed for mutagenesis. Restriction endonuclease sites used for cloning are underlined.