Figure 3: Possible orientations of pKOV Kan mORF51 cointegrants as seen by AgeI digest. (a) AgeI restriction map of the HVS genome. The restriction site marked in red is introduced when homologous recombination occurs between the pKOV Kan mORF51 plasmid and the complementary sequence in the viral genome. This introduced restriction site can then be used for identification of cointegrants. (b) Possible orientations of cointegrants. Depending on which homology region the reaction occurs (either 5 or 3 to the mutated ORF51 gene in pKOV Kan mORF51), cointegrants can be in one of two orientations. The mutated region containing the introduced AgeI site is shown in red. The pKOV Kan-mORF51 DNA is shown in blue, and the HVS DNA is shown in grey. The two orientations of cointegrant can be detected upon AgeI digestion analysis due to a 7 kb difference in the size of the ~50 kb fragment (see table). The larger ~80 kb fragment contains the terminal repeat (TR) region of the genome so is not suitable to detect this small difference as the number of TRs is not fixed.