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Two-hybrid systems are extremely powerful methods for detecting protein:protein interactions in vivo. The basis of two-hybrid systems is the modular domains found in some transcription factors: a DNA-binding domain, which binds to a specific DNA sequence, and a transcriptional activation domain, which interacts with the basal transcriptional machinery. A transcriptional activation domain in association with a DNA-binding domain will promote the assembly of RNA polymerase II complexes at the TATA box and increase transcription. In the CheckMate Mammalian Two-Hybrid System the DNA-binding domain and the transcriptional activation domain, produced by separate plasmids, are closely associated when one protein (X) fused to a DNA-binding domain interacts with a second protein (Y) fused to a transcriptional activation domain. In this system, interaction between proteins X and Y results in transcription of a reporter gene.

The CheckMate/Flexi Vector Mammalian Two-Hybrid System provides a means to confirm, validate and study suspected interactions between two proteins or domains and can be used to generate stable cell lines for cell-based assays. Developed primarily for mammalian proteins of interest, the system can allow protein expression and post-translational modifications in an environment mimicking the native cell milieu. It is patterned on the yeast two-hybrid system with one protein of interest (X) fused to a DNA-binding domain and the other protein (Y) fused to a transcriptional activation domain. The system relies upon three plasmids that are co-transfected into mammalian cells, each plasmid having unique features. This system differs from the original CheckMate Mammalian Two-Hybrid System in that the vectors are compatible with the Flexi Vector System, which allows directional cloning and rapid, efficient and high-fidelity transfer of protein coding regions between a variety of Flexi Vectors.

Co-transfection of mammalian cells with the pAdVAntage Vector enhances transient protein expression in a variety of cell types by increasing translation initiation. Transfection of mammalian cells with an expression vector often results in suboptimal expression of the protein of interest. Double-stranded RNA (dsRNA) generated during transfection is thought to activate the dsRNA-activated inhibitor (DAI), one of several enzymes involved in the host cell's antiviral defense system. DAI phosphorylates the translation initiation factor eIF-2, halting translation and therefore protein production. However, DAI translation inhibition can be overcome with the adenoviral Virus Associated I RNA (VAI RNA) produced by RNA polymerase III following co-transfection with the pAdVAntage Vector. The VAI RNA binds to DAI, preventing its activation, thereby allowing translation and protein expression.

The pCI-neo Mammalian Expression Vector carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells. This vector also contains the neomycin phosphotransferase gene, a selectable marker for mammalian cells. The pCI-neo Vector can be used for transient or stable expression by selecting transfected cells with the antibiotic G-418.

The pCMVTNT and pTNT Vectors are designed for convenient expression of cloned genes in vitro or in vivo. SP6 and T7 promoters allow expression from SP6- or T7-based coupled in vitro transcription/translation systems. The presence of RNA phage promoters also allows highly efficient synthesis of RNA in vitro. Both vectors contain a 5´ beta-globin leader sequence and synthetic poly(A)30 tail, which have been shown to enhance expression of certain genes. For in vivo expression, the pCMVTNT Vector contains a CMV enhancer/promoter region, which allows strong constitutive expression in many cell types.

This vector is designed for expression of N-terminal-tagged HaloTag fusion proteins in mammalian cells. Once expressed, the HaloTag fusion protein may be used for cell imaging of protein localization or trafficking in conjunction with the fluorescent HaloTag Ligands. In addition, the HaloTag fusion protein can be purified or pulled down as a complex with its protein partners. We offer two types of HaloTag fusion vectors to accommodate your cloning preferences: pHT Vector Series: Simple Multiple Cloning Site (MCS) plasmids for traditional cloning. pF Vector Series: Flexi Vector Cloning System---a directional cloning method for protein-coding sequences. It is based on two rare-cutting restriction enzymes, SgfI and PmeI, and provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between a variety of Flexi Vectors without the need to resequence.

These vectors are designed for expression of N-terminal-tagged HaloTag fusion proteins in mammalian cells. Once expressed, the HaloTag fusion protein may be used for cell imaging of protein localization or trafficking in conjunction with the fluorescent HaloTag Ligands. In addition, the HaloTag fusion protein can be purified or pulled down as a complex with its protein partners. We offer two types of HaloTag fusion vectors to accommodate your cloning preferences: pHT Vector Series: Simple Multiple Cloning Site (MCS) plasmids for traditional cloning. pF Vector Series: Flexi Vector Cloning System---a directional cloning method for protein-coding sequences. It is based on two rare-cutting restriction enzymes, SgfI and PmeI, and provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between a variety of Flexi Vectors without the need to resequence.

The pTARGET Mammalian Expression Vector System is a convenient system for cloning PCR products and expressing cloned PCR products in mammalian cells. Prepare the vector by digesting with EcoRV and adding a 3' terminal thymidine to each end. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid in two ways. First, the overhangs prevent recircularization of the vector; second, they provide a compatible overhang for PCR products generated by thermostable polymerases that add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of amplified fragments. This vector contains a modified coding sequence of the alpha-peptide of beta-galactosidase, which allows recombinants to be selected using blue/white screening, and carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells.