In addition to the original pcDNA™5/FRT vector, four more Flp-In™ expression vectors-pcDNA5/FRT/V5-His-TOPO®, pSecTag/FRT/V5-His-TOPO®, pEF5/FRT/V5-DEST™, and pEF5/FRT/V5-D-TOPO® (Figure 1)-are available for cloning and expressing in the Flp-In™ System. These vectors offer a variety of cloning options and different promoter types. Vectors include the following features:
• Flp Recombinase Target (FRT) site for efficient integration into Flp-In™ Cell Lines • Hygromycin resistance gene for convenient selection of integrants • C-terminal V5 tag for easy detection of fusion proteins with the Anti-V5 Antibody • 6xHis tag (pcDNA5/FRT/V5-His-TOPO® and pSecTag/FRT/V5-His-TOPO® vectors only) for rapid purification of fusion proteins on nickel-chelating resin
All vectors feature either the high-level CMV or EF-1α promoter. In addition pSecTag/FRT/V5-His-TOPO® contains the Igκ leader sequence for secretion of recombinant protein.
Entry Options pcDNA™5/FRT is suitable for restriction digest-mediated cloning. The other four Flp-In™ expression vectors offer two time-saving options for cloning into a Flp-In™ expression vector: • Five-minute TOPO® Cloning
The pcDNA™5/FRT/V5-His and pSecTag/FRT/V5-His TOPO® TA Expression Kits offer five-minute cloning of Taq-amplified PCR products directly into a Flp-In™ expression vector. The pEF5/FRT/V5 Directional TOPO® Expression Kit allows five-minute directional cloning of PCR product generated with a proofreading enzyme. Using Directional TOPO® Cloning, >90% of the resulting clones will be in the correct orientation for expression, reducing time spent colony screening. • Easy Recombination into a Gateway® vector
The pEF5/FRT/V5-DEST™ vector is compatible with Gateway® Technology* for easy transfer of your gene of interest into different vectors or host systems. The fast, efficient Gateway® recombination reaction can be simultaneously performed with multiple destination vectors, saving you time when working with different systems.
Choice of Promoters Flp-In™ expression vectors are available with the CMV or EF-1α promoter. The pEF5/FRT/V5-DEST™ and pEF5/FRT/V5-D-TOPO® vectors contain the human EF-1É promoter for driving expression of the gene of interest. The EF-1α promoter expresses in a wide range of mammalian cell types, including those where the CMV promoter expression is absent or inconsistent. This promoter may be more appropriate for long-term gene expression in some cell types and is recommended for expression in the BHK and 3T3 pre-made Flp-In™ Cell Lines. The pcDNA5/FRT/V5-His-TOPO® and pSecTag/FRT/V5-His-TOPO® vectors carry the CMV promoter for high-level constitutive expression of your gene of interest in most cell types.
For Research Use Only. Not for use in diagnostic procedures.
Constitutive or Inducible System:
Selection Agent (Eukaryotic):
Contents & storage
The pcDNA™5/FRT/V5-His TOPO™ TA Expression Kit, pSecTag/FRT/V5-His TOPO™ TA Expression Kit, and the pEF5/FRT/V5 Directional TOPO™ Expression Kit contain two boxes. The TOPO™ Cloning box contains all of the reagents necessary to clone a Taq-amplified PCR product (TOPO™ TA) or a PCR product generated with a proofreading enzyme (Directional TOPO™), including 200 ng of topoisomerase Iactivated vector, sterile water, dNTPs, 10X PCR buffer, salt solution, control template and primers, primers for sequencing, and an expression control plasmid. Store at -20°C. The One Shot™ Competent Cell box contains all of the reagents necessary for transformation, including twenty-one 50-µl aliquots of One Shot™ TOP10 Chemically Competent E. coli, S.O.C. medium, and pUC19 supercoiled control plasmid. Store at -80°C. The pEF5/FRT/V5-DEST™ vector contains 6 µg of lyophilized vector and a control plasmid. Store at -20°C once resuspended. All reagents are guaranteed stable for 6 months when properly stored.