pAd328

* Armed conditionally replicative adenoviruses (CRAds)

* Promoter insertion in front of the E1a TATA Box

* Transgene insertion into the E3 region (2.7 kb deletion)

* AdenoQuick cloning system

pAd328 is a 39 kb plasmid that contains the sequences encompassing bp 466-right end (1.3–100 mu) of the Ad5 genome, with a 2.7 kb deletion in the E3 region. 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 promoter and the E3 region. The right ITR is flanked by PacI and SwaI sites. pAd328 is used in combination with the shuttle vectors pE1.2 and pE3.1 to construct conditionally replicative adenoviruses (CRAds) containing a heterologous promoter in front of the E1a TATA box, and a transgene in place of the E3 region. The maximum combined promoter/transgene capacity is 4.4 kb.