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Vectors: PCR Cloning Change

Name Description Part Number
pTARGET Mammalian Expression Vector System Open/Close Add
Clone PCR products with T overhangs
CMV promoter for robust, consistent expression
Neomycin marker for selection of stable transfectants

The pTARGET Mammalian Expression Vector System is a convenient system for cloning PCR products and expressing cloned PCR products in mammalian cells. Prepare the vector by digesting with EcoRV and adding 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. This vector contains a modified coding sequence of the alpha-peptide of beta-galactosidase, which allows recombinants to be selected using blue/white screening, and carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells.

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pGEM-T Vector Systems Open/Close Add
Insert excision with a BstZI single digest
Ligation can be completed in 1 hour at room temperature
Available with or without competent cells

The pGEM-T Vector Systems are convenient systems to clone PCR products. The pGEM-T Vector is prepared by cutting the pGEM-5Zf(+) Vector with EcoRV and adding a 3' terminal thymidine to both ends. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for ligation of PCR products generated by thermostable polymerases that add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of amplified fragments. The multiple cloning site is flanked by recognition sites for the restriction enzyme BstZI, allowing release of the insert by a single-enzyme digestion. Alternatively, a double digestion may be used to release the insert from the vector. The pGEM-T Vector System II contains JM109 Competent Cells in addition to all of the pGEM-T Vector System I components.

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pGEM-T Easy Vector Systems Open/Close Add
Three options for removing the insert with a single digest (BstZI, EcoRI or NotI )
Ligation can be completed in 1 hour at room temperature
Available with or without competent cells

The pGEM-T Easy Vector Systems are convenient systems to clone PCR products. They offer all of the advantages of the pGEM-T Vector Systems with the added convenience of recognition sites for EcoRI and NotI flanking the insertion site. Thus, several options exist to remove the desired insert DNA with a single restriction digestion. The pGEM-T Easy Vector System II contains JM109 Competent Cells in addition to all of the pGEM-T Easy Vector System I components.

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