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Convenient system for cloning and expression of PCR products in mammalian cells.
The pTARGET Mammalian Expression Vector System is a convenient system for cloning PCR products and for expressing cloned PCR products in mammalian cells. The vector is prepared by digestion with EcoRV followed by addition of a 3' terminal thymidine to each end. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid in two ways. First, the overhangs prevent recircularization of the vector; second, they provide a compatible overhang for PCR products generated by thermostable polymerases that add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of amplified fragments. The pTARGET Vector also contains a modified coding sequence of the alpha-peptide of beta-galactosidase, which allows recombinants to be selected using blue/white screening. The pTARGET Vector carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells. This vector also contains the neomycin phosphotransferase gene, a selectable marker for mammalian cells. The pTARGET Vector can be used for transient expression or stable expression by selecting transfected cells with the antibiotic G-418.