Replication-deficient Ad5 with CMV-βGal cassette in place of the E1 region

Ad5.CMVβGal is an Ad5-based adenovirus containing a 4.3 kb-long LacZ expression cassette in place of the E1 region, oriented from right to left relative to the adenovirus genome.  The E. coli β-galactosidase coding sequence is fused to a SV40 nuclear localization signal  (GPKKKRKVGS) and is under the control of a human cytomegalovirus (CMV) promoter and a mouse protamine 1 polyadenylation signal.  Both E1 (3.1 kb) and E3 (2.7 kb) regions are deleted.  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.