orientation the asp718 site close to the sv40 derived poly signal was designated piclhaw plasmids piclhac and piclhaw are represented in fig1 and 17 plasmid picli was created from plasmid picl by insertion of the sali sgrai fragment from picl containing the ad5 itr into the asp718 site of picl the 194 bp sali sgrai fragment was isolated from picl and the cohesive ends were converted to blunt ends using e coli dna polymerase i klenow fragment and dntp 39 s the asp718 cohesive ends were converted to blunt ends by treatment with mungbean nuclease by ligation clones were generated that contain the itr in the asp718 site of plasmid picl a clone that contained the itr fragment in the correct orientation was designated picli see fig1 recombinant adenovirus was constructed according to the method described in european patent application 95202213 two components are required to generate a recombinant adenovirus first an adaptor plasmid containing the left terminus of the adenovirus genome containing the itr and the packaging signal an expression cassette with the gene of interest and a portion of the adenovirus genome which can be used for homologous recombination second adenovirus dna is needed for recombination with the aforementioned adaptor plasmid in the case of ad cmv hctk the plasmid pcmv tk was used.