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 查看更多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.
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For Research Use Only. Not for use in diagnostic procedures.
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