Schematic representation of the construction of a recombinant adenovirus that expresses a synthetic DNA encoding for a polyepitope protein which contains 46 HCMV T-cell epitopes (see box and Table 1).

<p>Each of the alternate epitope sequences are shown in bold letters. The DNA sequence encoding this polyepitope protein was constructed using overlapping epitope sequence specific primers (referred to as CMV1 to CMV20) as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003256#s4" target="_blank">Material and Methods</a>” section. This synthetic insert was first cloned into a pBluescript II KS⁺ phagemid, prior to cloning into the pShuttle vector. After amplification in <i>E.coli</i>, the expression cassette from pShuttle was excised and ligated into the Ad5F35 expression vector. Following linearization of the DNA using Pac I restriction enzyme, the recombinant Ad5F35 vector was packaged into infectious adenovirus by transfecting HEK 293 cells, and recombinant adenovirus (referred to as Ad-CMVpoly) was harvested from transfected cells by repeated freeze-thawing cycles.