Figure 4: AgeI restriction analysis of cointegrants formed by homologous recombination. The mutated version of the ORF51 gene contains an AgeI restriction site. This extra restriction site can be used as a marker for the integration of the pKOV Kan mORF51 vector into the HVS genome. Pulse field gel electrophoresis of possible cointegrant clones 3, 4, 8, and 9 reveals successful integration, as the ~130 kb band seen in HVS-GFP-BAC is cleaved into 2 smaller bands (indicated by arrows) due to the introduced restriction site. The 2 different orientations of pKOV Kan mORF51 can be visualised as clones 3 and 4 show a restriction pattern with bands at 45 kb and ~90 kb, whereas clones 8 and 9 have bands at 52 kb and ~85 kb. This 7 kb difference in the restriction site corresponds to the length of pKOV Kan mORF51.