pTargeT™ Mammalian Expression Vector System

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, ...

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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 version of the coding sequence of the α-peptide of β-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.

Features - Benefits

• Simple PCR Cloning: "T" overhangs permit direct ligation of PCR products generated by thermostable enzymes such as Taq DNA polymerase.
• Strong, Constitutive Expression: The CMV enhancer/promoter region allows strong, constitutive expression in many cell types. In transgenic mice, expression of the chloramphenicol acetyltransferase (CAT) gene under the regulation of the CMV enhancer/promoter was observed in 24 of the 28 tissues examined. The vector is maintained as an episome in cells expressing the SV40 large T antigen, leading to even higher levels of expression.
• Blue/White Screening: Allows easy identification of recombinant clones. A single digest removes the insert DNA.
• Stable Transfectants: Select for stable transfectants using the neomycin phosphotransferase gene.

Applications

For more information, see the Protocols & Applications Guide.

References

1. Mezei, L.M. and Storts, D.R. (1994) In: PCR Technology: Current Innovations, Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL, 21.
2. Robles, J. and Doers, M. (1994) Promega Notes 45, 19–20.
3. Clark, J.M. (1988) Nucl. Acids Res. 16, 9677–86.
4. Newton, C.R. and Graham, A. (1994) In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK, 13.
5. Schmidt, E.V. et al. (1990) Mol. Cell. Biol. 10, 4406–11.

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