This vector uses the human U6 promoter to expres a small hairpin RNA sequence that can silence gene expression. A short cDNA that includes a sequence from the target gene is ligated into the insertion site adjacent to the promoter.  The cDNA is constructed by annealing compementary oligonucleotides to one another.  Each oligonucleotide is designed with a 4-base overhang at its 5กฏ-end.  The overhangs complement vector sequences that remain after digestion with a pair of restriction enzymes.  The other features of the plasmid facilitate its rapid conversion to an adenovirus vector using the SpeAd procedure.

shRNA:   5'-actagtgAAAAAgacgcgt-3'         3'-tgatcacTTTTTctgcgca-5'             Spe I          Mlu I   (shown with a stop sequence that will be replaced with the cDNA insert)