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.
Specifications
Constitutive or Inducible System
Constitutive
Delivery Type
Transfection
Promoter
CMV
Product Type
Flp-In System Expression Vector
Selection Agent (Eukaryotic)
None
Protein Tag
Untagged
Cloning Method
TOPO, Gateway
Vector
pOG44
Product Line
Flp-In™
For Use With (Application)
Targeted Integration
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.