The pGreenPuro™ Vector is designed to express a single-stranded shRNA sequence with a fold-back stem-loop structure (also known as a “hairpin”) from a RNA polymerase III H1 promoter .The hairpin-type siRNA (shRNA) template oligonucleotides need to be cloned into unique BamHI/EcoRI sites located just downstream of an H1 promoter. The pGreenPuro™ vector needs to be linearized by restriction digest with BamHI and EcoRI, and purified to remove the stuffer fragment. When linearized, the vector contains two unique 5’ overhangs to facilitate directional cloning of shRNA template oligos with minimal self-ligation background . When the shRNA construct is expressed from constitutive H1 promoter and terminated with the TTTTT sequence, the shRNA transcript folds into the hairpin structure, which is recognized by the DICER enzyme, cleaved to form a functional ds siRNA and transferred to a RISC complex for selective digestion of complementary target mRNAs .Two PCR primers are designed for regions flanking the shRNA insert in order to provide a simple way for screening of plasmid clones for the
