Table 1.

Primer pairs for assembly of the TGEV infectious constructa

PrimerSequencePosition in genomeOrientationPurpose
TGEV 5′ T75′-GTCGGCCTCTTAATACGACTCACTATAGACTTTTAAAGTAAAGTGAG-3′1–19+Insert T7 start TGEV A, 5′ end
TGEV A 3,6425′-TGTTGAAGAAATCAAAGGCCTG-3′3621–3642Remove T7 stop
TGEV A 3,6215′-CAGGCCTTTGATTTCTTCAAC-3′3621–3641+Remove T7 stop, overlap RT-PCR
              T
TGEV A 6,1805′-CTTGGAGATGTTGAAAATCAGC-3′6180–6201TGEV A, 3′ end
TGEV B1 6,1345′-TAAAGTCTGCAGTCTGTGGC-3′6115–6134+TGEV B1, 5′ end
TGEV B1 9,9575′-TAATCCAAGTGAATGGTGTGTAC-3′9935–9957TGEV B1, 3′ end, insert BstXI site
TGEV B2 9,9395′-ACACCATTCACTTGGATTAATCC-3′9939–9961+TGEV B2, 5′ end, insert BstXI site
     C
TGEV B2 11,3425′-GTAGCCGATGCGGCTGGAATG-3′11342–11362TGEV B2, 3′ end
TGEV C 11,3455′-TCCAGCCGCATCGGCTACAAG-3′11345–11365+TGEV C, 5′ end, insert BglI site
      T     A
TGEV C 13,6055′-GTGCAAAGAAGAAGTGTTTTAATG-3′13605–13628Remove T7 stop
TGEV C 13,6095′-AAAACACTTCTTCTTTGCACAGG-3′13609–13631+Remove T7 stop, overlap RT-PCR
      T  T
TGEV C 16,5805′-TGTGCCAAGAAGGCCTTGACAAC-3′16580–16602TGEV C, 3′ end
TGEV DE 16,5855′-CAAGGCCTTCTTGGCACATAATC-3′16585–16607+TGEV DE, 5′ end, insert BglI site
         T  A
TGEV DE 3,1335′-GTCTAGCCTGCACGGCTACTGC-3′23475–23496TGEV DE, 3′ end
TGEV F 3,1125′-GCAGTAGCCGTGCAGGCTAGAC 23475–23496+TGEV F, 5′ end, insert BglI site
        A  T
TGEV 3′ end5′-NNNNNNGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTTTGGTGTATCACTATCAAAAGG-3′3′ end, poly(A) tailTGEV F 3′ end, Poly(A) tail,NotI
  • a When a primer sequence differs from the wild-type sequence, the wild-type nucleotide is shown below the sequence. Restriction sites are shown in boldface, and the T7 start site in underlined. Sequences are from references 3, 11,17, and 33.