FIG. 2.

Shuttling mutated hexons into the complete AdC68 genome. The R1 and 2 and R3 to 5 variable regions are labeled and represented as arrows (orange) below a larger arrow identifying the hexon gene (orange) in the plasmid. The size of these arrows varies according to the size of the plasmid, and in the larger plasmids (i.e., pAdC68mutGFP), the R1 and 2 region is represented as a small blue box rather than an arrow. Nonunique restriction sites are labeled in black and unique sites in red. In all plasmids, f₁ ORI, APr, CMV, and GFP (green) identify the origin of replication, ampicillin resistance, CMV promoter, and green fluorescent protein genes, respectively. The mutated hexon gene was excised from a mutagenesis vector (pHexon Mut) with the restriction enzymes ClaI and NheI and cloned into these same sites in a shuttle vector (pAdC68Shuttle) for final ligation into a plasmid containing the complete coding sequence for AdC68. In the last step of the cloning strategy, the 25-kb SpeI fragment from pAdC68Shuttle, containing the hexon mutation, was ligated with the SpeI-digested pC68GFP backbone to generate a pPAN9CMVGFP (AdC68mutGFP) mutant vector.