FIG. 1.
Construction of a VP26-GFP fusion protein. The 5.2-kb EcoRI L fragment of HSV-1 strain KOS was cloned into pUC19. The EcoRI L fragment contains UL35 (VP26) and the C-terminal-encoding sequences of the UL34 and UL36 genes (14). A shortened version of the EcoRI L clone spanning the EcoRI and NotI restriction sites was used for subsequent manipulations. An XhoI restriction site which spans residues 5 and 7 of the UL35 ORF was created by overlap extension PCR assays (8, 12). This plasmid was cleaved with XhoI, and an oligonucleotide duplex specifying NcoI and BsrGI restriction sites was annealed at this position. The GFP ORF derived from pEGFP-N1 (Clontech) as an NcoI-BsrGI fragment was cloned into this plasmid to generate pK26GFP. UL, unique long; US, unique short.