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BacMam pCMV-Dest Vector (Invitrogen™)

The BacMam pCMV-Dest Vector combines Gateway® cloning and BacMam gene expression technologies for easy recombination-based cloning and baculovirus-based expression of a target gene in variety of cell types. With BacMam technology, a modified insect cell virus (baculovirus) is used as a vehicle to efficiently deliver and transiently express genes in mammalian cells with minimum effort and toxicity. This technology allows for control over the level of expression through the increase or decrease of viral particle concentration. When combined with Gateway® cloning technology, a variety of sizes of target genes can be cloned and expressed. Sizes ranging from an insert for RNAi to a large gene with introns (38 kb) have been cloned and expressed successfully using the BacMam pCMV-Dest Vector. This plasmid accommodates various cloning schemes, including Gateway® , Seamless Assembly, and traditional cloning using restriction enzymes.

The BacMam pCMV-Dest Vector contains a CMV promoter to drive constitutive expression of the target gene. It also contains VSV-G elements, which enable baculovirus to have high transduction efficiency, and a WPRE element, which elongates transient expression in mammalian systems. To facilitate successful cloning, AmpR and gentamicin have been incorporated for positive selection, as well as Cm(R) (chloramphenicol resistance gene) for negative selection. Upon successful cloning, the region containing the CmR is replaced by the insert, selecting against any bacteria that still have a CmR-containing region.

pLenti6/UbC/V5-DEST™ Gateway™ Vector (Invitrogen™)

Invitrogen™'s pLenti6⁄UbC⁄V5-DEST™ Gateway® Vector Kit is part of our ViraPower™ UbC Lentiviral Gateway® Expression Kit (catalog K4990-00).

The pLenti6⁄UbC⁄V5-DEST™ Gateway® Vector is a Gateway®-adapted ViraPower™ lentiviral expression vector for lentiviral-based expression of a target gene in dividing and non-dividing mammalian cells. The vector has the UbC promoter for driving constitutive but physiological levels of expression of the target gene and the blasticidin selection marker for stable selection in mammalian cells.

Advantages
• Stable expression
• Long-term experiments
• Accurate titer of functional virus
• Flexible and versatile Gateway® recombination cloning technology

Key Features
• UbC promoter
• V5 epitope tag at C terminus
• Blasticidin selection

Kit includes
• A lacZ vector as a positive control, pLenti6⁄UbC⁄V5-GW⁄lacZ
• Stbl3™ competent cells

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

pTracer™-SV40 Mammalian Expression Vector (Invitrogen™)

Three pTracer™ mammalian expression vectors are available that express GFP fused to the selectable marker Zeocin™ . Each vector uses a different set of promoters to express the gene of interest and the Cycle 3 GFP-Zeocin™ fusion.

All three pTracer™ vectors have the following features:

• Cycle 3 GFP-Zeocin™ fusion for selection in mammalian cell lines
• BGH polyA signal and transcription termination sequences to enhance mRNA stability

The pTracer™-SV40 vector offers:
• SV40 promoter for high-level constitutive expression of your gene of interest
• CMV promoter for high-level constitutive expression of the Cycle 3 GFP-Zeocin™ fusion
• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the SV40 large T antigen

The pTracer™-CMV2 vector offers:
• CMV promoter for high-level constitutive expression of your gene of interest
• EF-1α promoter for high-level constitutive expression of the Cycle 3 GFP-Zeocin™ fusion
• Ampicillin resistance gene for selection in E. coli

The pTracer™-EF vectors offer:
• EF-1α promoter for high-level constitutive expression of your gene of interest
• CMV promoter for high-level constitutive expression of the Cycle 3 GFP-Zeocin™ fusion
• C-terminal V5 epitope tag for simple detection of recombinant fusion proteins with an Anti-V5 Antibody
• C-terminal 6xHis tag for rapid purification on nickel-chelating resin and simple detection with an Anti-His(C-term) Antibody
• Multiple cloning site supplied in three reading frames to simplify cloning in frame with the C-terminal tag

Gateway™ pT-Rex™-DEST30 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.

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

This pcDNA™3.1(-) vector is designed for high-level, constitutive expression in a variety of mammalian cell lines. It contains a Geneticin® 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

pT7CFE1-NFtag Vector for Mammalian Cell-Free Protein Expression (Thermo Scientific™)

Thermo Scientific pT7CFE1-NFtag is a cloning plasmid optimized to use with the Thermo Scientific 1-Step Human In Vitro Protein Expression System for in vitro translation (IVT) of tagged fusion proteins. pT7CFE1-NFtag Vector is available with single affinity Ftag tag at the N-terminus to facilitate protein purification and detection.

Features of pT7CFE1:
• EMCV IRES at the 5' UTR promotes high-level translation of mRNAs
• MCS accommodates gene insertion via ten different restriction sites: Msc1, Nde1, BamH1, EcoR1, EcoRV, Pac1, Pst1, Sac1, Sal1, Not1 and Xho1
• Poly A sequence in the 3' region promotes mRNA stabilization and protection from nucleases
• T7 terminator ensures synthesis of accurate size mRNA transcripts
• Plasmid linearization may be accomplished with restriction sites between Poly A sequence and the T7 terminator region

pT7CFE1 Expression Vectors contain the Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) element that is critical for high levels of cap-independent protein expression in the Human In Vitro Translation System. Each vector features a highly-compatible multiple cloning site (MCS) to facilitate easy insertion of protein coding sequences into and between vectors. The pT7CFE1 Vector is available with single or tandem affinity tags at the N- or C- terminus to facilitate protein purification and detection. The pT7CFE Vectors are suitable for insertion of cloned genes, cDNAs, ORFs or PCR products for in vitro transcription and translation. Custom cloning services are also available.

More Product Data
Choosing a vector and purification method for in vitro protein expression

Related Products
pT7CFE1-Cftag Vector for Mammalian Cell-Free Protein Expression
pT7CFE1-CGST-HA-His Vector for Mammalian Cell-Free Protein Expression
pT7CFE1-NHis-GST Vector for Mammalian Cell-Free Protein Expression
pT7CFE1-NHis-GST-CHA Vector for Mammalian Cell-Free Protein Expression

Champion™ pET300/NT-DEST and pET301/CT-DEST Gateway™ Vector Kit (Invitrogen™)

The Champion™ pET300/NT-DEST and pET301/CT-DEST Gateway® Vector Kit is designed for rapid cloning with a Gateway® entry clone and subsequent high-level prokaryotic expression controlled by the strong bacteriophage T7 promoter. In addition to the T7 promoter, each vector contains only the necessary functional elements and an N- or C-terminal 6xHis tag (pET300/NT-DEST and pET301/CT-DEST, respectively) for convenient purification and detection (Figure 1). The vector kit is ideal for structural biologists who desire no or minimal modifications to their protein of interest. To maximize expression, use with MagicMedia™ E. coli Expression Medium.



Contents and Storage:
The Champion™ pET300/NT-DEST and pET301/CT-DEST Gateway® Vector Kit includes 6 µg each of pET300/NT-DEST and pET301/CT-DEST vectors and 10 µg of control vector. Store at -20“C. Guaranteed stable for 6 months when properly stored.

TA Cloning™ Kit, with pCR™2.1 Vector and One Shot™ TOP10 Chemically Competent E. coli (Invitrogen™)

The TA Cloning® Kit with pCR™2.1 vector provides a quick, one-step cloning strategy for directly inserting a Taq-amplified PCR product into a plasmid vector. The TA Cloning® Kit uses the pCR™2.1 cloning vector and ExpressLink™ T4 DNA Ligase to generate a ligation product in a fifteen-minute, room-temperature ligation step. Reactions typically yield >80% recombinants containing inserts.

Features of the TA Cloning® Kit with pCR™2.1 vector:
Fast & convenient—15-minute, room-temperature ligation
Efficient—blue/white screening and >80% clones with correct insert
Flexible—choice of kanamycin or ampicillin resistance for flexible antibiotic selection
Hassle-free—eliminates any enzymatic modifications of the PCR product
Streamlined—does not require the use of PCR primers that contain restriction sites

The pCR™2.1 vector provides:
• 3'-T overhangs for direct ligation of Taq-amplified PCR products
• T7 promoter for in vitro RNA transcription and sequencing
• A versatile polylinker with flanking EcoR I sites for easy excision of inserts
• M13 forward and reverse primer sites for sequencing

How TA Cloning® Works
Taq polymerase has a non-template-dependent activity that adds a single deoxyadenosine (A) to the 3' ends of PCR products. The linearized vector supplied in this kit has single 3' deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

Kit Configurations
The TA Cloning® Kit is offered in a variety of configurations: without competent cells (K2020-20 and K2020-40), with One Shot® INVF' Chemically Competent E. coli (K2000-01 and K2000-40), with One Shot® TOP10F' Chemically Competent E. coli (K2030-01 and K2030-40), and with One Shot® TOP10 Chemically Competent E. coli (K2040-01 and K2040-40) in 20- and 40-reaction kit sizes.

PichiaPink™ Vector Kit (Invitrogen™)

The PichiaPink™ Vector Kit contains the pPINK-HC and pPINK-LC vectors for use with the PichiaPink™ Yeast Expression System. The pPINK vectors are built around the ADE2 gene for complementing the ade2 deletion in the PichiaPink™ strains. You can order the PichiaPink™ Vector Kit to refill your PichiaPink™ Yeast Expression System Kits.

The PichiaPink™ Vector Kit comes with:
•     pPINK-LC vector (low-copy number) (20 µg)
•     pPINK-HC vector (high-copy number) (20 µg)(1)
•     5’AOX1 sequencing primer (20 µg)
•     3’CYC3 sequencing primer (20 µg)
•     One Shot® TOP10 Electrocomp™ E. coli (Cat. No. C404052)

Two Vector Options
The PichiaPink™ Vector Kit comes with two vectors: pPINK-LC (low-copy number) and pPINK-HC (high-copy number). You can choose the vector that works better at producing your protein. Both vectors use the methanol-induced AOX1 promoter to express your protein.

Both pPINK-LC and pPINK-HC vectors are compatible with the PichiaPink™ Secretion Signal Set. Using a 3-way ligation, you can add one of 8 secretion sequences to your gene. The pPINK vectors express proteins intracellularly by default.

Why Choose the PichiaPink™ Yeast Expression System?
The PichiaPink™ Yeast Expression System is based on the yeast Pichia pastoris. Advantages of Pichia pastoris include rapid growth, well-defined genetic background, simple media formulation, and easy handling. For over 30 years, Pichia pastoris has been used by labs around the world for producing hundreds of different proteins from many species including human (2,3). The PichiaPink™ Yeast Expression System allows convenient and cost-effective protein production from small to large scales.

For information on obtaining a commercial-use license for the PichiaPink™Yeast Expression System, please inquire at outlicensing@lifetech.com.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Related Links
Download the pPINK-LC plasmid map (PDF).
Download the pPINK-HC plasmid map (PDF).
Learn more about the PichiaPink™ Yeast Expression System.
Learn more about our other protein expression systems.

References

1. Du, et al. (2012) Bioengineered Bugs 3:1, 32-37.
2. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000 Jan;24(1):45-66. [PubMed]
3. Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol. 2002 Aug;13(4):329-32. [PubMed]

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.

pOG44 Flp-Recombinase Expression Vector (Invitrogen™)

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 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.

pEF1α Tluc16-DD Vector for Luciferase Assays (Thermo Scientific™)

The Thermo Scientific™ pEF1α Tluc16 Vector encodes the smallest known luciferase, TurboLuc™16 (Tluc16) luciferase, as a reporter under a modified version of the Elongated Factor 1α (EF1α) promoter for constitutive Tluc16 luciferase expression in mammalian cells.

• Intracellular Tluc16 luciferase gene, optimized for high expression in mammalian systems
• Truncated EF1α promoter for constitutive Tluc16 luciferase expression

Tluc16 luciferase is a 16 kDa, novel, intracellular luciferase derived from the marine copedod Metridia luciferase family. The wild-type luciferase has been modified to reduce its size, increase its brightness, and enable its intracellular expression. The Tluc16 luciferase activity can be measured using the Thermo Scientific TurboLuc™ One-Step Glow Assay Kit.

Gateway™ pDONR™221 Vector (Invitrogen™)

Gateway® Technology enables rapid cloning of one or more genes into virtually any protein expression system. Once you have an entry clone, you can recombine your gene of interest into a variety of expression vectors adapted for use in Gateway® Technology. The PCR product is directionally cloned with efficiencies typically >99%. The pDONR™221 vector has a pUC origin for high plasmid yields and universal M13 sequencing sites for ease of use.

Gateway™ pENTR™ 3C Dual Selection Vector (Invitrogen™)

Gateway® entry vectors are designed to clone DNA sequences using restriction endonucleases and ligase to create a Gateway® entry clone. The resulting entry clone is ready for recombination with a destination vector to create an expression clone. New: pENTR™ Dual Selection vectors!

The Gateway® entry vectors (Table 1) offer the following:
• attL1 and attL2 sites for site-specific recombination of the entry clone with a Gateway® destination vector to ensure cloning of the gene of interest in the correct orientation for expression
• Kozak consensus sequence for efficient translation initiation in eukaryotic systems
• Ribosome binding site for efficient translation initiation in prokaryotic systems (pENTR™ 1A Dual Selection, pENTR™3C Dual Selection, and pENTR™11 Dual Selection vectors only)
• rrnB transcription termination sequences to prevent basal expression of the PCR product of interest in E. coli
• pUC origin for high-copy replication and maintenance of the plasmid in E. coli
• Kanamycin resistance gene for selection in E. coli
• The ccdB⁄chloramphenicol fusion gene located between the two attL sites for
o negative selection and
o Chloramphenicol selection in E. coli
• Kanamycin resistance gene for selection in E. coli

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.