The pcDNA™ vectors are designed for high-level, constitutive expression in a variety of mammalian cell lines. The pcDNA3.2/GW/D-TOPO vector offers the following key features:

•Cytomegalovirus (CMV) promoter for high-level expression
•Adapted for Directional TOPO® Cloning, enabling you to use proofreading polymerases and to clone your PCR products in a specific orientation
•Neomycin resistance gene for stable selection
•C-terminal V5 tag for easy detection
•Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli

TOPO® Cloning
Using restriction enzymes to clone your gene into an expression vector often forces you to compromise the final sequence of your insert (Figure 1A), especially when there are no useful restriction sites close to your genes coding sequence. This may result in suboptimal spacing of expression elements or incorporation of non-native amino acid residues, which can reduce your expression levels and/or cause the production of non-functional protein.

In addition to being a more effective way to clone, TOPO® Cloning eliminates these potential expression problems. TOPO® Expression Vectors enable you to insert the exact DNA sequence you require simply by performing PCR with appropriately designed primers. Your PCR product is cloned at a high efficiency in only five minutes into a topoisomerase I-activated expression vector. The resulting recombinant expression vector contains your exact DNA sequence without any non-coding regions (Figure 1B).

Many of Life Technologies' powerful expression vectors are available adapted for one-step TOPO® cloning and expression of PCR products. In addition, several expression vectors are now adapted for Directional TOPO® Cloning, enabling you to use proofreading polymerases and to clone your PCR products in a specific orientation.