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pcDNA™4/TO/myc-His A, B, & C Mammalian Expression Vectors (Invitrogen™)

A Tetracycline-Regulated Expression System without Viral Transactivators
The T-REx™ System yields higher levels of induced expression than any other regulated mammalian expression system. It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known (1,2).

Specific Activation
The T-REx™ System uses a repressor mechanism that blocks transcription from the powerful CMV promoter in the absence of tetracycline. Because the T-REx™ System elements do not use viral transactivators, you can achieve high-level expression from the complete CMV promoter without secondary, non-specific activation of host genes.

The T-REx™ Mechanism
The T-REx™ transcriptional control elements are illustrated in Figure 1. Two tetracycline operator sequences (TetO2) have been inserted between the TATA box of the CMV promoter and the transcriptional start site. The TetO2 sequence itself has no effect on expression. When the tetracycline repressor protein (TR) is present, it effectively binds the TetO2 sites and blocks transcription initiation. Tetracycline added to the culture medium binds to, and changes the conformation of, the TR protein. This change causes the TR protein to release the TetO2 sites, derepressing transcription from the CMV promoter. The result is high-level expression of the gene of interest (Figure 2). Expression levels can be modulated based on the tetracycline concentration and can be induced to levels that are achieved with constitutive CMV expression vectors.
T-REx™ is a powerful inducible mammalian expression system that allows you to regulate expression from the complete human cytomegalovirus (CMV) enhancer-promoter. T-REx™inducible expression vectors offer the following features:

• Complete CMV enhancer-promoter sequence containing two copies of the tetracycline operator TetO2 sequence for high-level regulated expression
• Zeocin™ or hygromycin resistance gene for effective selection of stable mammalian cell lines
• Large multiple cloning site to simplify cloning

In addition, pcDNA™4/TO/myc-His offers a c-myc epitope for rapid detection of the recombinant protein with an Anti-myc Antibody and a polyhistidine (6xHis) sequence for simple purification of the recombinant protein with nickel-chelating resin and detection with Anti- His(C-term) Antibody.

The regulatory vector, pcDNA™6/TR, is provided for high-level expression of the tetracycline repressor (TR) protein. This vector expresses the Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express the TR protein.

Vivid Colors™ pcDNA™6.2/EmGFP-Bsd/V5-DEST Vector (Invitrogen™)

The Vivid Colors™ pcDNA™ 6.2/EmGFP-Bsd/V5-DEST vector allows you to rapidly clone your gene using Gateway® technology and simultaneously express it with Emerald Green Fluorescent Protein (EmGFP) for easy identification of transfected cells. The EmGFP is derived from well-characterized fluorescent proteins of the jellyfish Aequorea victoria (1, 2) and has been humanized for optimal mammalian expression (3) (Figure 1). If cloned with the TAG (amber) codon, your gene will be expressed in its native configuration and fully compatible with Tag-On-Demand" technology. If cloned without a stop codon, a V5-epitope tag will be fused to the carboxy-terminal end of your protein of interest. In either case, expression of EmGFP provides a bright fluorescent marker to easily identify cells co-expressing your protein. This marker allows you to focus your functional analysis on specific cells, or to sort and enrich for transfected cells in a derived population.

The Vivid Colors™ pcDNA™ 6.2/EmGFP-Bsd/V5-DEST vector provides:

• PGK promoter for high-level expression of EmGFP fused to the Blasticidin (bsd)-resistance marker
• CMV promoter for high-level expression of your protein
• V5-epitope tag option to fuse to the C-terminal end of your protein, if desired

pcDNA™6.2/GW/D-TOPO™ Expression Kit (Invitrogen™)

The pcDNA™ vectors are designed for high-level, constitutive expression in a variety of mammalian cell lines. The pcDNA6.2/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
•Blasticidin resistance gene for efficient 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 our 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.

pcDNA™3.1/nV5-DEST Mammalian Expression Vector (Invitrogen™)

To fit all of your expression needs, Invitrogen offers state-of-the-art Gateway® destination vectors for expression in E. coli, insect, yeast, or mammalian cells, as well as for production of native protein or N- or C-terminal fusion proteins. All Gateway® destination vectors have attR sites for recombination with any attL-flanked fragment, regardless of whether it is an entry clone or an Ultimate™ RF Clone. The following table lists the wide range of destination vectors available.

Additional materials required, available separately: Gateway® entry clone, Gateway® LR Clonase® enzyme mix, and reaction buffer.

pRABBIT IgG IRES-EmGFP Positive Control Vector (Gibco™)

The pRABBIT IgG IRES-EmGFP Positive Control Vector is a mammalian expression control that expresses a complete, full-length rabbit IgG and EmGFP. It can be used as a positive expression control for transient expression systems such as the Expi293 Expression System, the ExpiCHO Expression System, the FreeStyle 293 Expression System, and the FreeStyle CHO Expression System.

Features of the pRABBIT IgG IRES-EmGFP Positive Control Vector include:
• A mix of pcDNA3.4 plasmids containing the coding sequences for EmGFP and the heavy and light chains of rabbit IgG
• Optimized mix of IgG light and heavy chain transfection-grade plasmids in a single, ready-to-use tube
• Expression of rabbit IgG at 450–500 mg/L in Expi293F cells
• Sufficient material for transfection of up to 150 mL of suspension culture in the Expi293, ExpiCHO, and FreeStyle systems

This control is included in the Expi293 GnTI-, Expi293 Inducible, and Expi293 Inducible GnTI- expression system kits.

pLenti6.2/V5-DEST™ Gateway™ Vector (Invitrogen™)

The pLenti6.2⁄V5-DEST™ Gateway® Vector is a Gateway®-adapted ViraPower™ II lentiviral expression vector for lentiviral-based expression of a target gene in dividing and non-dividing mammalian cells. The vector has the CMV promoter for driving constitutive expression of the target gene and the PGK promoter for driving long-term, persistent expression of the Blasticidin stable selection marker.

Advantages
• Stable expression
• Long-term experiments
• High-throughput screening
• Accurate titer of functional virus (using Blasticidin method)
• Flexible and versatile Gateway® recombination cloning technology

Key Features
• Human cytomegalovirus (CMV) immediate early promoter to control high-level expression of the gene of interest
• PKG Promoter for expression of Blasticidin selection marker

Kit includes
• pLenti6.2⁄V5-DEST™ Gateway® Vector box
• One Shot® Stbl3™ chemically competent E. coli (C7373-03)

Related SKUs
• 293FT Cell Line (R70007; R70007)
• ViraPower™ Lentiviral Support Kit (K4970-00)
• ViraPower™ Lentiviral Packaging Mix (K4975-00)
• Lipofectamine® 2000 (11668-019; 11668-027)

For research use only. Not intended for any therapeutic or diagnostic use.

pJTI™ R4 EXP CMV-TO EmGFP pA Vector

The pJTI™ R4 Exp CMV-TO EmGFP pA vector is a positive control vector for assessing retargeting efficiency when retargeting a Jump-In™ T-REx™ parental cell line. When co-transfected with the integrase vector (pJTI™ R4 Int vector included in the Jump-In™ parental kits) and after antibiotic selection, the EmGFP can be inducibly expressed with doxycycline and the successfully retargeted cells will fluoresce green.

Ensure the Success of Your Jump-In™ T-REx™ Retargeting Reactions
Successful retargeting of Jump-In™ T-REx™ parental cell lines like the Jump-In™ T-REx™ HEK293 Kit is dependent on a variety of factors, such as:

• Transfection efficiency
• Cell confluency
• Antibiotic selection conditions
• Quality and concentration of DNA
• Retargeting vector to integrase vector ratio

We strongly recommend including a positive control retargeting reaction using the pJTI™ R4 Exp CMV-TO EmGFP pA vector in your Jump-In™ experiment, along with negative controls (no plasmid DNA, no integrase vector), so you can easily visualize the results and optimize the retargeting conditions.

pcDNA™5/TO Mammalian Expression Vector (Gibco™)

pcDNA5/TO is a 5.7 kb expression vector designed for use with the T-REx System (Cat. Nos. K1020-01 and K1020-02) and Expi293 Inducible platform (Cat. Nos. A39251 and A39252). It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known.

The pcDNA5/TO vector allows tetracycline-regulated expression of the gene of interest in mammalian host cells expressing the Tet repressor (TetR) from the pcDNA6/TR vector (Catalog no. V1025-20).

pcDNA™6.2/V5-PL-DEST Mammalian Expression Vector (Invitrogen™)

The pcDNA™ vectors are designed for high-level, constitutive expression in a variety of mammalian cell lines. The pcDNA6.2/V5-pL-DEST vector offers the following key features:

•Promoterless version of the pcDNA™6.2⁄V5-DEST vector (cat. no. 12489027)
attR sites for Gateway® cloning
•Compatible with MultiSite Gateway® Pro kits (e.g. cat. no. 12537100)
•C-terminal V5 tag for easy detection
•Blasticidin resistance gene for efficient stable selection
•Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli

Gateway® Cloning
To fit all of your expression needs, Invitrogen offers state-of-the-art Gateway® destination vectors for expression in E. coli, insect, yeast, or mammalian cells, as well as for production of native protein or N- or C-terminal fusion proteins. All Gateway® destination vectors have attR sites for recombination with any attL-flanked fragment, regardless of whether it is an entry clone or an Ultimate™ ORF Clone. The following table lists a variety of available destination vectors.

Additional materials required, available separately: Gateway® entry clone, appropriate Gateway® LR Clonase® enzyme mix, and reaction buffer.

pLenti6.2-GW/EmGFP Expression Control Vector (Invitrogen™)

The Vivid Colors™ pLenti6.2-GW⁄EmGFP Expression Control Vector is a ViraPower™ positive control lentiviral vector containing Emerald Green Fluorescent Protein (EmGFP). It is designed for use with the ViraPower™ Lentiviral Expression Systems as a positive control to enable the detection of EmGFP fluorescence following transfection in 293FT cells, as a titer control to produce an EmGFP-expressing lentivirus stock and as a transduction control following transduction in both dividing and non-dividing mammalian cells. The vector has the CMV promoter for driving constitutive expression of EmGFP and the PGK promoter for driving long-term, persistent expression of the Blasticidin stable selection marker. This is not a cloning vector.

Advantages
• Optimization of viral transduction efficiency
• Optimization of 293FT Transfection
• 2 day titer of functional virus using EmGFP

Key Features
• Human cytomegalovirus (CMV) immediate early promoter to control high-level expression of the EmGFP
• PKG Promoter for expression of Blasticidin selection marker

Kit Includes
pLenti6.2-GW⁄EmGFP Vector

Related SKUs
• 293FT Cell Line (R70007; R70007)
• ViraPower™ Lentiviral Support Kit (K4970-00)
• ViraPower™ Lentiviral Packaging Mix (K4975-00)
• Lipofectamine® 2000 (11668-019; 11668-027)

For research use only. Not intended for any therapeutic or diagnostic use.

pAd/PL-DEST™ Gateway™ Vector Kit (Invitrogen™)

The pAd⁄PL-DEST™ Gateway® Vector Kit contains the Gateway®-adapted ViraPower™ adenoviral expression vector, pAd⁄PL-DEST® vector for easy recombination-based cloning and adenoviral-based, transient expression of a target gene in dividing and non-dividing mammalian cells. The vector allows generation of an adenovirus containing the target gene where expression is driven by a promoter of choice. Alternatively, the vector may also be used to express small RNA molecules from their appropriate promoters.

Advantages
• High efficiency and rapid recombination cloning
• Produces high titer adenoviral stocks
• Efficient delivery of the gene to dividing and non-dividing mammalian cells in vitro or in vivo
• Allows gene of interest to be controlled by a promoter of choice
• Produces replication-incompetent virus for enhanced biosafety of the system
• Amenable for use in high-throughput applications

Key Features
• Gateway® Technology for efficient and rapid cloning
• Promoterless vector that allows gene of interest to be controlled by a promoter of choice
• Human Ad5 sequences (ΔE3) and Viral Inverted Terminal Repeats (ITRs) for packaging of the expression construct into virions
• Ampicillin selection marker

Kit includes
• pAd⁄PL-DEST™ Vector
• pAd⁄CMV⁄V5-GW⁄lacZ control plasmid

For research use only. Not intended for any therapeutic or diagnostic use.

pcDNA™3.1/Zeo (-) Mammalian Expression Vector (Invitrogen™)

This pcDNA™3.1/Zeo(-) vector is designed for high-level, constitutive expression in a variety of mammalian cell lines. It contains a Zeocin™ selectable marker and a reverse-orientation multiple cloning site.

The pcDNA™3.1 Expression Vector Family
Three untagged versions of pcDNA™3.1 (available separately), each with a different selectable marker (Geneticin®, Zeocin™, or Hygromycin), are for use alone or in co-transfections. All three vectors offer the following features:
• Cytomegalovirus (CMV) enhancer-promoter for high-level expression
• Large multiple cloning site in either forward (+) or reverse (-) orientations
• Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence for enhanced mRNA stability
• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (i.e., COS-1 and COS-7)
• Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli

pZeoSV2(+) Vector Kit (Invitrogen™)

pZeoSV2(+) and (-) are 3.5-kb vectors designed for high-level, constitutive expression in mammalian cell lines that express the SV40 large T antigen. The Zeocin™ resistance gene provides fast and efficient selection in E. coliand mammalian cells that do not express the SV40 large T antigen. The vectors have the following features which make them efficient, easy-to-use tools:

• SV40 enhancer-promoter and origin for high-level, constitutive expression and replication in mammalian cells
• BGH polyA signal for efficient processing of mRNA transcripts
• Multiple cloning site in the forward (+) and reverse (-) orientations for easier cloning
• Small size for efficient cloning
• f1 origin for the rescue of single-strand DNA (sense strand)
• pUC origin of replication for growth in E. coli

Expression of the Zeocin™ resistance gene is driven by the CMV promoter in mammalian cell lines and by the synthetic EM-7 promoter in E. coli. pZeoSV2 can be used in transient expression assays as well as to create stable cell lines.

pcDNA™6.2-DEST Mammalian Expression Vector (Invitrogen™)

The pcDNA™ vectors are designed for high-level, constitutive expression in a variety of mammalian cell lines. The pcDNA6.2/V5-DEST vector offers the following key features:

•Cytomegalovirus (CMV) promoter for high-level expression
attR sites for Gateway® cloning, enabling recombination with attL-flanked fragments
•C-terminal V5 tag for easy detection
•Blasticidin resistance gene for efficient stable selection
•Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli

Gateway® Cloning
To fit all of your expression needs, Invitrogen offers state-of-the-art Gateway® destination vectors for expression in E. coli, insect, yeast, or mammalian cells, as well as for production of native protein or N- or C-terminal fusion proteins. All Gateway® destination vectors have attR sites for recombination with any attL-flanked fragment, regardless of whether it is an entry clone or an Ultimate™ RF Clone. The following table lists a variety of available destination vectors.

Additional materials required, available separately: Gateway® entry clone, appropriate Gateway® LR Clonase® enzyme mix, and reaction buffer.

pcDNA™3.1/myc-His A, B, & C Mammalian Expression Vectors (Invitrogen™)

All pcDNA™ vectors contain a strong promoter for high-level expression in mammalian cells, a choice of selection marker for generating stable cell lines, and an epitope tag for easy detection with a monoclonal antibody and rapid purification on nickel-chelating resin. Each vector is available in three reading frames to simplify cloning in-frame with the fusion tag.