TABLE 1.
Primers used to generate the mutant full-length cDNAs and the pCIneo expression constructs containing ORFs 2a and 2b of PRRSV LV
| Primer designationa | Primer sequence (5′→3′)b | Nucleotide positionc | Purpose |
|---|---|---|---|
| LV183 (+) | GCCGTGAATTCGGGGTGTAGCAATGGGG | 11779 | KO2a |
| LV25 (−) | AATCGGATCCTGGTCAGCTCGAATGATGTG | 12529 | KO2a and KO2b |
| LV189A (+) | GCCGTGAATTCGGGGTGATGCAGTGGGGTCAC | 11779 | KO2b |
| LV250 (−) | ACTGGGCCCTCAGCTCGAATGATG | 12531 | KO3 |
| LV253 (−) | ACTGGGCCCTTATCGTGATGTACTGGGGAGTAC | 13178 | KO4 |
| LV90 (+) | TCGACTTGTCGCTAGTATGC | 10798 | KO3, KO4, and R− |
| LV186 (+) | TGTTCGCCATTCTCTTGGCAATCTGAGATGT | 13472 | KO5 |
| LV38 (−) | AATCGGATCCTCTGGTTTTTACCGGCC | 14600 | KO5 |
| LV96 (+) | CCAACTGCAGACCGGATGTCC | 12025 | KO6 |
| LV187 (−) | ATCGTCTAGGCCTCCCACTGCTCAGCC | 14078 | KO6 |
| LV407 (−) | AGGCGGCAGCCGGGGCTTGCCTAGCTCTACCTGC | 11737 | R− |
| LV408 (−) | ATGTTAACGACGCGTCGTCACGGCACTTGCCCAGGCGGCAGCCG | 11759 | R− |
| LV271 (+) | GCTCTAGAGCCGCCGCCATGCAATGGGGTCACTGTGG | 11796 | pCIneo-ORF2a; incorporation of XbaI plus consensus Kozak sequence |
| LV278 (−) | ATAGTTTAGCGGCCGCTCAGCTCGAATGATGTGTTGC | 12525 | pCIneo-ORF2a; incorporation of NotI |
| LV272 (+) | GCTCTAGAGCCGCCGCCATGGGGTCACTGTGGAG | 11801 | pCIneo-ORF2b; incorporation of XbaI plus consensus Kozak sequence |
| LV279 (−) | ATAGTTTAGCGGCCGCTCATAGGACCTTCGATAGTTCG | 12013 | pCIneo-ORF2b; incorporation of NotI |
a
Positive-sense primers and negative-sense primers are marked by a + and −, respectively.
b
The restriction sites are indicated by single underlining, and the consensus Kozak sequence is indicated by double underlining. The mutated translation initiation sites are indicated in boldface type.
c
The position of each primer with respect to the nucleotide sequence of PRRSV LV (GenBank accession no. M96262).