TABLE 2.
PCR primers used for generation of BHV-1 UL49.5 cysteine residue mutants and for colony identification
| Primer function and name | Sequence (5′ to 3′)a |
|---|---|
| Mutagenesis | |
| C42S F | 5′-GATGCGGCGCGAGGGGGCAATGGACTTTTGGAGCGCAGGCtcgTACGCGCGCGGGGTGCCGCTAGGATGACGACGATAAGTAGGG-3′ |
| C42S R | 5′-GGGCCTGCGGTGGCTCCGAGAGCGGCACCCCGCGCGCGTAcgaGCCTGCGCTCCAAAAGTCCACAACCAATTAACCAATTCTGATTAG-3′ |
| C78S F | 5′-CGCGGTAATGGTCGCCGTGGCCCTGTACGCGTACGGGCTTtcgTTTAGGCTCATGGGCGCCAGAGGATGACGACGATAAGTAGGG-3′ |
| C78S R | 5′-ACTCCTTTTTATTGGGCCCGCTGGCGCCCATGAGCCTAAAcgaAAGCCCGTACGCGTACAGGGCAACCAATTAACCAATTCTGATTAG-3′ |
| C78S CT- null F | 5′-CGCGGTAATGGTCGCCGTGGCCCTGTACGCGTACGGGCTTtcgTTTTAAGCGCCAGCGGGCCCAGGATGACGACGATAAGTAGGG-3′ |
| C78S CT-null R | 5′-CCCCGCGACTCCTTTTTATTGGGCCCGCTGGCGCTTAAAAcgaAAGCCCGTACGCGTACAGGGCAACCAATTAACCAATTCTGATTAG-3′ |
| Verificationb | |
| UL49.5 F | 5′-AGAGCGCCAGCGAGTCGGGCTC-3′ |
| UL49.5 R | 5′-AACCGGGCCATGGCAAGCGAGTC-3′ |
a
BHV-1 UL49.5-specific sequences are shown in bold uppercase letters; the italicized sequences of forward (F) and reverse (R) primers, respectively, are complementary to each other in inverse orientation. Lowercase nucleotides code for serine instead of cysteine. Underlining indicates the pEPkan-S-specific sequences.
b
Primers used to PCR amplify the UL49.5 ORF and its downstream sequences for identification of BHV-1 UL49.5 BAC mutants from the selected kanamycin-sensitive colonies for sequencing.