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. 2003 Jan;77(1):696–700. doi: 10.1128/JVI.77.1.696-700.2003

TABLE 1.

Construction of C2 expression cassettesa

Construct Vector Primer pair (5′ region/ 3′ region) Cloning sites Mutation
pBac/C2 pFastBacHTa PP141/PP142 NcoI, EcoRI Wild type
pBac/C2-C36R pFastBacHTa PP141/PP142 NcoI, EcoRI Cys36→Arg
pBac/C2-C38N pFastBacHTa PP141/PP143 NcoI, BamHI Cys38→Asn
pBac/C2-C46I pFastBacHTa PP141/PP143 NcoI, BamHI Cys46→Ile
pEHT/C2 pEHISTEV PP141/PP142 NcoI, EcoRI Wild type
pEHT/C2-C36R pEHISTEV PP141/PP142 NcoI, EcoRI Cys36→Arg
pEHT/C2-C38N pEHISTEV PP141/PP143 NcoI, BamHI Cys38→Asn
pEHT/C2-C46I pEHISTEV PP141/PP143 NcoI, BamHI Cys46→Ile
a

The TYLCV-C C2 gene and mutant derivatives were PCR amplified using PVX/C2, PVX/C2-C36R, PVX/C2-C38N, and PVX/C2-C46I (37) as DNA templates together with primer PP141 (cctgtatcATGaGATCTTCGTCTCCCTC) and either PP142 (ccgaatTcAAATACTCTTAAGAAATGCGAGGTC) or PP143 (ccggatccTAAATACTCTTAAGAAATGCGAGGTC) (introduced restriction endonuclease sites are underlined and modified nucleotides are lowercase). PCR products were digested with BspHI plus EcoRI or BamHI and were cloned into the NcoI and EcoRI or NcoI and BamHI sites of pEHISTEV (a modified version of pET28a [unpublished data]) and pFastBacHTa (Invitrogen Life Technologies) to produce vectors for C2 protein expression in E. coli and insect cells, respectively.