Science Catalog 2012 Life Cell Signaling pTARGET™ Mammalian Expression Vector System Product pTARGET™ Mammalian Expression Vector System For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pTARGET™ Mammalian Expression Vector System is a convenient system for cloning PCR products and for expression of 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 certain thermostable polymerases. These polymerases often 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) immediateearly 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 for stable expression by selecting transfected cells with the antibiotic G-418. Features: • 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 the easy identification of recombinant clones. A single digest removes the insert DNA. • Stable Transfectants: Select for stable transfectants using the neomycin phosphotransferase gene. Storage Conditions: Store competent cells at –70°C; store all other components at –20°C or –70°C. Protocol Part# pTARGET™ Mammalian Expression Vector System Technical Manual TM044 BglII 5665 ori Ampr Enhancer/Promoter Intron CMV pTARGET™ Vector (5670bp) Synthetic poly(A) Neo SgfI 664 I-PpoI 851 T T SV40 Late poly (A) fl ori SV40 Enhancer/ EarlyPromoter lacZ T7 EcoRI BamHI NheI XhoI MluI SmaI KpnI SalI AccI NotI 1250 1256 1264 1270 1276 T overhangs EcoRI lacZ 1293 1301 1303 1304 1311 1318 Size Cat.# Price (Fr) 20 reactions A1410 850.00 pTARGET™ Sequencing Primer Product pTARGET™ Sequencing Primer For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pTARGET™ Sequencing Primer is designed for sequencing inserts cloned into the pTARGET™ Mammalian Expression Vector (Cat.# A1410). The sequencing primer hybridizes to the region of the lacZ gene from nucleotides 1367–1344 on the pTARGET™ Vector. The primer can be used only for sequencing inserts cloned into the pTARGET™ Vector. The primer sequence is not a binding site for any RNA polymerases and cannot be used to generate in vitro transcripts. The sequence of the pTARGET™ Sequencing Primer is 5´-d(TTACGCCAAGTTAT TTAGGTGACA)-3´. The primer is supplied at a concentration of 10ng/μl (1.25pmol/μl) in sterile water. Storage Conditions: Store at –20°C. Size Cat.# Price (Fr) 2 µg Q4461 135.00 222 For complete and up-to-date product information visit: www.promega.com/catalog 1505VA07_6A Pagina 225
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