pAd363 is a 35.5 kb plasmid that contains the sequence encompassing bp 3504-right end (9.8–100 mu) of the Ad5 genome. The two SfiI sites naturally present in WT Ad5 DNA were mutated by substituting A for G and C at positions 16291 and 16294 in the Ad5 genome, and C and G for respectively G and C at positions 23001 and 23004 in the Ad5 genome, introducing silent mutations in the adenovirus pVII and DNA-binding protein coding sequences. Two pairs of SfiI sites that allow for directional cloning replace the E1 and E3 regions. The E3 region contains a 2.7 kb deletion that corresponds to bp 28137 to 30819 in the Ad5 genome. The E4 region contains a 1.2 kb deletion that encompasses ORF 1-4, and corresponds to bp 34119-35355 in the WT Ad5 genome. The right ITR is flanked by PacI and SwaI sites. pAd363 is used in combination with the shuttle plasmids pE1.2 and pE3.1 to construct “second-generation” bipartite replication-deficient adenoviruses containing transgenes in place of the E1 and E3 regions. These recombinant adenoviruses are able to grow in 293 cells without E4 trans-complementation. The maximum combined transgene capacity is 8.7 kb.