TABLE 1.
Primers used in this study
| Primer | Sequence (5′→3′)a |
|---|---|
| H262A_SD_F | GGACTACGCGTTTGAAgcCGTTGTTTATGGTAGTTTTAACC |
| H262A_SD_R | GGTTAAAACTACCATAAACAACGGCTTCAAACGCGTAGTCC |
| G275A_SD_F | CCAGAAGATTATTGGAGcTTTGCATTTGCTTATTGGCTTAGCCC |
| G275A_SD_R | GGGCTAAGCCAATAAGCAAATGCAAAGCTCCAATAATCTTCTGG |
| H277A_SD_F | CCAGAAGATTATTGGAGGTTTGgcTTTGCTTATTGGCTTAGCCC |
| H277A_SD_R | GGGCTAAGCCAATAAGCAAAGCCAAACCTCCAATAATCTTCTGG |
| D324A_SD_F | GTAAGAGTGTGTGCACTGTTATTGCTTTATTGTTAGATGATTTTGTGG |
| D324A_SD_R | CCACAAAATCATCTAACAATAAAGCAATAACAGTGCACACACTCTTAC |
| Oligonucleotide 35-5_R | GCT CGG ATC CAC TAG TAA CGG CCG CCA GTG |
| Oligonucleotide 28-1_F | GTA TGT TAA CAA ACA TGC ATT CCA CAC C |
| MHVpcdna-ab | AAAAGGTACCATGGGTTTAGAAAATGTAGTG |
| MHVpcdna-bc | CGAAGGATCCTACAGATCCTCTTCAGAGATGAGTTTCTGCTCCTGCAAACGAGGATAGAAAGTCATGACCTTCTC |
| MHV 5′ 1-20 (+) | TATAAGAGTGATTGGCGTCC |
| 7059 (−) oligonucleotide 8d | CTGGTGTTACACAGGCAG |
| S_seq_R1 primere | ATTGCTGAGCGTAAACAACG |
| N (−) primerf | GGC ACT CCT TGT CCT TCT |
| B35For | TAG TAC TCT ACC TGG TTT T |
| 17TG | TTT TTT TTT TTT TTT TTG |
| GAPDH-F2 primer | ACCCAGAAGACTGTGGATGG |
| GAPDH-R2 primer | CACATTGGGGGTAGGAACAC |
a
Lowercase letters indicate mutations.
b
Sense primer containing a KpnI site, A at −3, a start codon (ATG), and G at +4 to replace S with G.
c
Antisense primer with a Myc tag, a stop codon (TGA), and BamHI site.
d
Corresponding to nt 611 to 628 in the MHV-A59 genome (GenBank accession number NC001846); used to specifically prime minus-strand sgRNA1.
e
Corresponding to nt 25320 to 25339 in the MHV-A59 genome; used to specifically prime minus-strand sgRNA3.
f
Corresponding to nt 29920 to 29937 in the MHV-A59 genome; used to specifically prime minus-strand sgRNA7.