pAd329 is a 41.7 kb plasmid that contains the sequences encompassing bp 466-right end (1.3–100 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. A pair of SfiI sites that allow for directional cloning replaces the E1 promoter. The E3 region is intact. The right ITR is flanked by PacI and SwaI sites. pAd329 is used in combination with shuttle plasmid pE1.1 and derivatives to construct conditionally replicative adenoviruses (CrAds) containing a heterologous promoter in front of the E1a TATA box. The maximum capacity for insertion is 1.9 kb.
