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Vectors: Mammalian Expression Change

Name Description Part Number
Regulated Mammalian Expression System Open/Close Add
Dose-response induction of protein expression
Rapid, responsive on/off protein expression switch

The Regulated Mammalian Expression System features low basal levels, robust and rapid induction, and downregulation of gene expression in mammalian cells. The Regulated Mammalian Expression System is based on a novel on/off switch that relies on the rapid and sensitive modulation by coumerin-related compounds of a chimeric transactivator protein. Nanomolar concentrations of the antibiotic coumermycin promote homodimerization of a chimeric transactivator that, in turn, binds to lambda operator sequences located upstream of a minimal promoter driving transcription of coding sequences for a protein of interest. The levels of protein expression can be regulated by adjusting the coumermycin concentration. More significantly, this expression can be promptly and effectively switched off by adding novobiocin, which acts as an antagonist by dissociating the dimerized transactivator protein. The protein coding region of interest is cloned into either the pF12A RM Flexi Vector or pF12K RM Flexi Vector, both of which are specially designed for Regulated Mammalian (RM) protein expression. These vectors incorporate regulatory promoter sequences upstream of the protein-coding region and are compatible with the Flexi Vector System. In transient transfection paradigms, the pF12A or pF12K RM Flexi Vector containing the protein-coding region of interest is co-transfected into mammalian cells together with the pReg neo Vector. The pReg neo Vector is designed to express a chimeric transactivator protein that interacts with the regulatory promoter region in the pF12A and pF12K RM Flexi Vectors in a regulated fashion in response to coumermycin and novobiocin. Additionally, the pReg neo Vector encodes a neomycin phosphotransferase gene that allows stable cell selection and generation with the antibiotic G-418.

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pTARGET Mammalian Expression Vector System Open/Close Add
Clone PCR products with T overhangs
CMV promoter for robust, consistent expression
Neomycin marker for selection of stable transfectants

The pTARGET Mammalian Expression Vector System is a convenient system for cloning PCR products and for expressing cloned PCR products in mammalian cells. The vector is prepared by digestion with EcoRV followed by addition of 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. The pTARGET Vector also contains a modified coding sequence of the alpha-peptide of beta-galactosidase, which allows recombinants to be selected using blue/white screening. The pTARGET 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 pTARGET Vector can be used for transient expression or stable expression by selecting transfected cells with the antibiotic G-418.

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pSI Mammalian Expression Vector Open/Close Add
SV40 enhancer/promoter region allows strong, constitutive expression in most cell lines
Multiple cloning sites exist for easy insertion of cDNA
Synthesize transcripts in vitro using the T7 RNA polymerase promoter or generate single-stranded DNA in E. coli using the f1 origin of replication

The pSI Mammalian Expression Vector promotes constitutive expression of cloned DNA inserts in mammalian cells. The major difference between the pCI and pSI Mammalian Expression Vectors is the enhancer/promoter region controlling expression of the inserted gene. The pSI Expression Vector contains the simian virus 40 (SV40) enhancer and early promoter region. This vector can be used for both transient and stable expression of genes. For stable expression, the pSI Vector must be co-transfected with an expression vector containing a selectable gene for mammalian cells.

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pFN28A and pFN28K HaloTag CMV-neo Flexi Vectors Open/Close Add
Enables constitutive protein expression in mammalian cells
Includes CMV immediate-early enhancer/promoter
Suited to stable or transient gene expression

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. Note: Flexi Vectors contain the lethal barnase gene to reduce background colonies without inserts during the subcloning procedure. Using the Flexi Vector Cloning System replaces the barnase gene with your insert. These vectors, as purchased, cannot be cultured in normal laboratory strains of E. coli without an insert. Find your gene, precloned, and experimentally validated at: www.promega.com/products/pm/halotag-technology/kazusa-collection

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pFN24A and pFN24K HaloTag CMVd3 Flexi Vectors Open/Close Add
Enables constitutive protein expression in mammalian cells
Includes a modified CMVd3 immediate-early enhancer/promoter
Suited to stable or transient gene expression

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. Note: Flexi Vectors contain the lethal barnase gene to reduce background colonies without inserts during the subcloning procedure. Using the Flexi Vector Cloning System replaces the barnase gene with your insert. These vectors, as purchased, cannot be cultured in normal laboratory strains of E. coli without an insert. Find your gene, precloned, and experimentally validated at: www.promega.com/products/pm/halotag-technology/kazusa-collection

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pFN23A and pFN23K HaloTag CMVd2 Flexi Vectors Open/Close Add
Enables constitutive protein expression in mammalian cells
Includes a modified CMVd2 immediate-early enhancer/promoter
Suited to stable or transient gene expression

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. Note: Flexi Vectors contain the lethal barnase gene to reduce background colonies without inserts during the subcloning procedure. Using the Flexi Vector Cloning System replaces the barnase gene with your insert. These vectors, as purchased, cannot be cultured in normal laboratory strains of E. coli without an insert. Find your gene, precloned, and experimentally validated at: www.promega.com/products/pm/halotag-technology/kazusa-collection

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pFN22A and pFN22K HaloTag CMVd1 Flexi Vectors Open/Close Add
Enables constitutive protein expression in mammalian cells
Includes a modified CMVd1 immediate-early enhancer/promoter
Suited to stable or transient gene expression

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. Note: Flexi Vectors contain the lethal barnase gene to reduce background colonies without inserts during the subcloning procedure. Using the Flexi Vector Cloning System replaces the barnase gene with your insert. These vectors, as purchased, cannot be cultured in normal laboratory strains of E. coli without an insert. Find your gene, precloned, and experimentally validated at: www.promega.com/products/pm/halotag-technology/kazusa-collection

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pFN21A and pFN21K HaloTag CMV Flexi Vectors Open/Close Add
Enables constitutive protein expression in mammalian cells
Includes CMV immediate-early enhancer/promoter
Suited to stable or transient gene expression

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. Note: Flexi Vectors contain the lethal barnase gene to reduce background colonies without inserts during the subcloning procedure. Using the Flexi Vector Cloning System replaces the barnase gene with your insert. These vectors, as purchased, cannot be cultured in normal laboratory strains of E. coli without an insert. Find your gene, precloned, and experimentally validated at: www.promega.com/products/pm/halotag-technology/kazusa-collection

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pCMVTNT and pTNT Vectors Open/Close Add
Express cloned genes in vitro or in vivo from SP6- or T7-based coupled in vitro transcription/translation systems

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.

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pCI-neo Mammalian Expression Vector Open/Close Add
Select for stably transfected cells with neomycin phosphotransferase gene
Increase the steady-state level of RNA with the late SV40 polyadenylation signal approximately fivefold more than the early SV40 polyadenylation signal
Synthesize transcripts in vitro using the T7 RNA polymerase promoter or generate ssDNA in E. coli using the f1 origin of replication

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.

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pAdVAntage Vector Open/Close Add
Increases translation initiation in cells transiently transfected with protein expression constructs
Can be used in a variety of cell lines

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.

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CheckMate/Flexi Vector Mammalian Two-Hybrid System Open/Close Add
Confirm, validate and study suspected protein or domain interactions
Generate stable cell lines for cell-based assays
Enables mammalian protein expression and post-translational modification studies in situ

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 also 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. The pFN10A (ACT) Flexi Vector contains a herpes simplex virus VP16 transcriptional activation domain upstream of the cloning site, and the pFN11A (BIND) Flexi Vector contains the yeast GAL4DNA-binding domain upstream of the cloning site. The pFN11A (BIND) Flexi Vector also expresses the Renilla reniformis luciferase under the control of the SV40 promoter, allowing normalization for differences in transfection efficiency. The third vector, pGL4.31[luc2P/GAL4UAS/Hygro] Vector, contains five GAL4 binding sites upstream of a minimal TATA box, which is upstream of a firefly luciferase gene that acts as a reporter for interactions between proteins X and Y. 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.

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CheckMate Mammalian Two-Hybrid System Open/Close Add
Mammalian system for studying interactions in cell line of choice
Study post-translational modifications including glycosylation, phosphorylation and acylation

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.

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