Figure 8. Construction of E3 shuttle plasmids and transgenes. In order to create replication-competent vectors, the nonessential E3 were genes replaced with either a HA1-con expression cassette or a GFPLuc fusion gene. The PCR products that were used to create the shuttle plasmids for Ad4 and Ad7 are shown (A). Both of the overlapping PCR products were designed to flank the E3 genes and contained unique AscI sites. The AscI flanked transgene expression cassettes are shown (B). The CMV expression cassettes were fused to a FRT flanked zeocin gene for selection of recombinants and ultimate removal by FLP recombinase.