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.