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ViraPower™ Lentiviral Gateway™ Expression Kit (Invitrogen™)

The ViraPower™ Lentiviral Gateway® Expression Kit includes all the components needed to generate lentivirus, including vector kit, 293FT cell line, and the support kit. It combines Invitrogen’s ViraPower™ Lentiviral and Gateway® technologies to facilitate easy recombination-based cloning and lentiviral-based expression of a target gene in dividing and non-dividing mammalian cells. The pLenti6⁄V5-DEST™ vector has the CMV promoter for driving constitutive expression of the target gene and the blasticidin selection marker for stable selection in mammalian cells.

Advantages
• Lentivirus based expression of a target gene in dividing and non-dividing mammalian cells

Key Features
• Flexible and versatile Gateway® recombination cloning technology
• Constitutive high expression with CMV promoter
• Blasticidin selection marker for stable selection
• C terminal V5 tag for quick detection

Kit includes
• pLenti6⁄V5-DEST™ vector
• ViraPower™ Bsd Lentiviral Support Kit (K4970-00)
• 293FT Cell Line (R70007)
• One Shot® Stbl3™ Chemically Competent E. coli (C7373-03)

Related SKUs
• pLenti6⁄V5™ Directional TOPO® Cloning Kit (K4955-10)
• ViraPower™ Lentiviral Directional TOPO® Expression Kit (K4950-00)
• ViraPower™ HiPerform™ Lentiviral Gateway® Expression Kit (K5330-00)
• ViraPower™ HiPerform™ Lentiviral TOPO® Expression Kit (K5310-00)

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

Antibody-Expressing Positive Control Vector (Gibco™)

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

Expresses rabbit IgG at ~250 mg/L in the Expi293™ Expression System
Contains sufficient material for transfection of up to 150 mL of suspension culture in the Expi293™ and FreeStyle™ systems
Protocols are provided to quantitate rabbit IgG expression from the Positive Control Vector

The Antibody-Expressing Positive Control Vector contains an optimized mix of IgG heavy chain and light chain genes. This control is included in the Expi293™ Expression System Kit.

Gateway™ pDEST™8 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.

ViraPower™ Bsd Lentiviral Support Kit (Invitrogen™)

The ViraPower™ Bsd Lentiviral Support Kit contains the necessary components to transfect 293FT cells to generate viral particles containing the target gene. The kit contains the ViraPower™ Lentiviral Packaging Mix, Lipofectamine® 2000 Reagent and blasticidin and is designed to be used with a ViraPower™ Lentiviral expression vector that has the blasticidin marker for stable selection. The kit is compatible with all of our ViraPower™ Lentiviral vectors, including the ViraPower™ HiPerform™ Lentiviral vectors.

Kit includes:
• ViraPower™ Lentiviral Packaging Mix
• Lipofectamine® 2000 (Cat # 11668027)
• Blasticidin powder (Cat # R21001)

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

CloneJET PCR Cloning Kit (Thermo Scientific™)

Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Any other blunt or sticky-end DNA fragment can be cloned. It is ideal for phosphorylated or non-phosphorylated DNA fragments. Ligation into the included positive selection vector takes only 5 minutes, yielding more than 99% recombinant clones. Blunt-ended PCR products generated with a proofreading enzyme are ligated directly into the cloning vector.

PCR products generated either with non-proofreading DNA polymerases or mixtures of DNA polymerases are blunted prior to ligation in 5 minutes with the thermostable DNA Blunting Enzyme provided with the kit. All common laboratory E. coli strains can be directly transformed with the ligation product.

Features

The CloneJET PCR Cloning Kit contains a novel, ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Recircularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme, which kills the host E. coli cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection.

For convenience in mapping and manipulation of the insert, the pJET1.2/blunt cloning vector multiple cloning site contains two BglII recognition sequences that flank the insertion site. In addition, the vector contains a T7 promoter for in vitro and in vivo transcription as well as sequencing of the insert.

Highlights

Fast—PCR cloning in only 5 minutes
Highest efficiency – > 99% of positive clones
No cloning background—positive selection vector
Versatile—ideal for blunt-end or sticky-end cloning
Economical—no expensive blue/white screening

Applications

• Cloning of blunt-end or 3'-dA tailed PCR products up to 10 kb
• Cloning of DNA fragments generated by restriction enzymes
• Sequencing of cloned DNA
in vitro and in vivo transcription of cloned inserts from the T7 promoter

Includes

• pJET1.2/blunt Cloning Vector
T4 DNA Ligase
• 2X Reaction Buffer
• DNA Blunting Enzyme
• pJET1.2 Forward Sequencing Primer (5'-CGACTCACTATAGGGAGAGCGGC-3')
• pJET1.2 Reverse Sequencing Primer (5'-AAGAACATCGATTTTCCATGGCAG-3')
• Control PCR Product
Water, nuclease-free
• Detailed Protocol

Prior to electroporation, always column-purify the ligation mixture using e.g. GeneJET PCR Purification Kit #K0701 or chloroform to extract it. Electroporation is inhibited by the presence of proteins and salts in the mixture.

Related Products
CloneJET PCR Cloning Kit
Fast DNA End Repair Kit

TOPO™ TA Cloning™ Kit, with pCR™2.1-TOPO™, One Shot Mach1™-T1R Chemically Competent E. coli, and PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™)

TOPO® TA Cloning® Kits are designed for cloning PCR products directly from a PCR reaction in just 5 minutes (1). They use a pCR™-TOPO® Vector with covalently bound topoisomerase I for fast cloning and recombinants. pCR™-TOPO® Vectors include:
• 3´-T overhangs for direct ligation of Taq-amplified PCR products
• Choice of T7 (pCR™2.1-TOPO®) or T7 and SP6 (pCR™II-TOPO®) promoters for in vitro RNA transcription and sequencing. Each vector also contains M13 forward and reverse primer sites for sequencing.
EcoR I sites flanking the PCR product insertion site for easy excision of inserts
• Kanamycin and ampicillin resistance genes for your choice of selection in E. coli
• Easy blue/white colony screening for selection of recombinants

TOPO® TA Cloning® Kits are available in combo kits combined with a PureLink™ Quick Plasmid Miniprep Kit (50 preps) for fast plasmid purification of your TOPO®-cloned inserts for downstream analysis.

ViraPower™ HiPerform™ Promoterless Gateway™ Expression System (Invitrogen™)

The ViraPower™ HiPerform™ Promoterless Gateway® Expression System includes all the components needed to generate lentivirus, including vector kit, 293FT cell line, and the support kit. This kit combines Invitrogen’s ViraPower™ HiPerform™ Lentiviral and MultiSite Gateway® technologies to facilitate easy recombination-based cloning and lentiviral-based high-level expression of a target gene from any promoter of choice in dividing and non-dividing mammalian cells. The promoterless pLenti6.4⁄R4R2⁄V5-DEST™ vector is equipped with two key genetic elements, making it a HiPerform™ vector: the Woodchuck Posttranscriptional Regulatory Element (WPRE) and the central Polypurine Tract (cPPT) sequence from the HIV-1 integrase gene to produce at least 4-fold increase in protein expression compared to vectors lacking these elements.

Advantages
• Generates replication-incompetent lentivirus for transducing dividing and non-dividing mammalian cells
• Easy, simultaneous, recombination-based cloning of multiple DNA fragments in a defined order and orientation using MultiSite Gateway® technology
• Expression of the target gene under the control of a promoter of choice
• Stable, long-term expression
• Enhanced protein expression, up to 4-fold or greater, compared to traditional lentiviral expression systems


Key Features
• WPRE from the woodchuck hepatitis virus, increases transgene expression and cPPT from the HIV-1 integrase gene, increases the copy number of lentivirus integrating into the host genome, thus increasing viral titer. WPRE and cPPT together produce at least a four-fold increase in protein expression in most cell types, compared to other vectors that do not contain these elements.
• Promoterless vector to express the target gene under the control of a promoter of choice
• Blasticidin selection marker for stable selection under control of PGK promoter for long-term, persistent expression

Kit includes
• ViraPower™ HiPerform™ Promoterless Gateway® Vector Kit (Cat # A11146)
• ViraPower™ Bsd Lentiviral Support Kit (Cat # K497000)
• 293FT Cell Line (Cat # R70007)
• Gateway® LR Clonase® II Plus Enzyme Mix (Cat # 12538120)

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

pTK-Gaussia-Dura Luc Vector for Luciferase Assays (Thermo Scientific™)

The Thermo Scientific pTK-Gaussia-Dura Luc vector is a derivative of pMCS-Gaussia-Dura Luc. It contains a mutant form of the Gaussia luciferase gene (conferring better bioluminescent signal stability than the native luciferase) under the control of the weak Herpes Simplex Virus (HSV) thymidine kinase (TK) promoter. This constitutive expression vector can be used as a normalization control to account for experimental variation in combination with other reporters.

Features of the pTK-Gaussia-Dura Luc vector:

• Weak (TK) constitutive promoter for co-transfection and normalization
• Naturally-secreting Gaussia-Dura luciferase gene, optimized for high expression and glow-stability in mammalian systems
• Multiple cloning site (MCS) provides versatility for transfer of regulatory elements from one plasmid to another
• Transcription termination site (Ter), Lac operator (Lac O1), and transcriptional pause site (TPS) used to minimize background by reducing transcriptional read-through
• Both puromycin (Pur) and ampicillin (Amp) markers for drug selection in mammalian and bacterial cells, respectively
• High-copy pUC bacterial DNA replication origin

Gaussia-Dura luciferase (approx. 20kDa) is a secreted protein that enables measurement of the reporter activity in media (for real-time assays) and in cell lysates. The parent pMCS vector contains a multiple cloning site for cloning a promoter to study its regulatory potential.

These vectors are subject to a limited use label license.

Related Products
pMCS-Gaussia-Dura Luc Vector for Luciferase Assays
pCMV-Gaussia-Dura Luc Vector for Luciferase Assays

pBAD/His Kit (Invitrogen™)

The pBAD/His Kit provides all of the necessary reagents to express your protein in a tightly regulated fashion. The vector pBAD/His allows you to express your protein with an N-terminal tag. The vector provides:

• The araBAD promoter for tightly regulated expression
• Translation initiation signals optimized for E. coliexpression
• N-terminal polyhistidine (6xHis) tag for purification with nickel-chelating resin and detection with an Anti-HisG Antibody
• N-terminal Xpress™ epitope for detection and analysis with an Anti-Xpress™ Antibody
• Enterokinase cleavage site for removing the N-terminal tag following purification

Three vectors are provided (A, B, and C). Each has the N-terminal tag in a different reading
frame relative to the multiple cloning site to simplify in-frame cloning of your gene.

Flp-In™ Complete System (Invitrogen™)

Our Flp-In™ Complete System allows integration and expression of your gene of interest in mammalian cells at a specific genomic location. The Flp-In System involves introduction of a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice. An expression vector containing your gene of interest is then integrated into the genome via Flp recombinase-mediated DNA recombination at the FRT site.

The major components of the Flp-In™ System include:

• A Flp-In. target site vector, pFRT⁄lacZeo, for generation of a host cell line containing an integrated FRT site
• An expression plasmid containing a FRT site linked to the hygromycin resistance gene for Flp recombinase-mediated integration and selection of a stable cell line expressing your gene of interest under the control of the human cytomegalovirus (CMV) immediate-early enhancer⁄promoter
• A Flp recombinase expression plasmid, pOG44, for expression of the Flp recombinase under the control of the human CMV promoter
• A control expression plasmid containing the chloramphenicol acetyl transferase (CAT) gene, which when cotransfected with pOG44 into your Flp-In. host cell line, expresses CAT

ViraPower™ Lentiviral Packaging Mix (Invitrogen™)

ViraPower™ Lentiviral Packaging Mix contains an optimized mixture of the three packaging plasmids, pLP1, pLP2, and pLP/VSVG. These plasmids supply the helper functions as well as structural and replication proteins in trans required to produce a recombinant lentivirus, containing your gene of interest, using our ViraPower™ Lentiviral Expression System.

The ViraPower™ Lentiviral Expression System
The ViraPower™ Lentiviral Expression System allows creation of a replication-incompetent, HIV-1–based lentivirus that is used to deliver and express your gene of interest in either dividing or non-dividing mammalian cells. The major components of the system include:

• An expression plasmid containing the gene of interest under the control of a choice of promoters, and elements that allow packaging of the construct into virions
• An optimized mix of the three packaging plasmids (pLP1, pLP2, and pLP/VSVG) that supply the structural and replication proteins in trans that are required to produce the lentivirus
• The 293FT cell line, which allows production of lentivirus following cotransfection of the expression plasmid and the plasmids in the packaging mix
• Control expression plasmid to optimize virus production and cell transduction, containing either the lacZ gene—which when packaged into virions and transduced into a mammalian cell line, expresses β-galactosidase (included with each expression vector), or the Emerald Green Fluorescent Protein (EmGFP) gene—which when packaged into virions and transduced into a mammalian cell line, expresses EmGFP.

aLICator LIC Cloning and Expression Kit 1 (untagged) (Thermo Scientific™)

Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.

The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.

Highlights

• High efficiency LIC cloning
• Tight control of gene expression
• High yield expression
• Versatile—tagged or untaged protein expression with tag removal option

Applications

• Directional PCR product cloning
• Tightly regulated protein expression
• Expression of toxic genes

Principle

The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.

The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.

Features

The system consists of four kits based on the pLATE series of bacterial expression vectors:

aLICator™ LIC Cloning and Expression Kit 1 - pLATE11 vector, untagged protein expression.
aLICator™ LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)—pLATE51 vector, N-terminal His-tag protein expression.
aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag)—pLATE31 vector, C-terminal His-tag protein expression.
aLICator™ LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)—pLATE 52 vector, N-terminal His-tag protein expression, WELQut cleavage.
aLICator™ LIC Cloning and Expression Set 1 (All-in-One/EK)—pLATE11, pLATE51 and pLATE31 vectors, choice of untagged, N- or C-terminal His-tag protein expression.
aLICator™ LIC Cloning and Expression Set 2 (All-in-One/WQ)—pLATE11, pLATE52, and pLATE31 vectors, choice of untagged, N- or C-terminal His tag protein expression, WELQut cleavage.

For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to clone into all three vectors and determine the most compatible vector for further research.

Related Products
aLICator LIC Cloning and Expression Kit 2 (N-terminal His-tag/EK)
aLICator LIC Cloning and Expression Kit 3 (C-terminal His-tag)
aLICator LIC Cloning and Expression Set 1 (All-in-One/EK)
aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ)
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ)

pPICZα A, B, & C Pichia Vectors (Invitrogen™)

pPICα A, B, and C vectors are 3.6 kb vectors used to express and secrete recombinant proteins in Pichia pastoris. Recombinant proteins are expressed as fusions to an N-terminal peptide encoding the Saccharomyces cerevisiae á-factor secretion signal. These vectors allow high-level, methanol inducible expression of the gene of interest in Pichia, and can be used in any Pichia strain including X-33, SMD1168H, and KM71H. pPICα vectors contain the following elements:


• Contains AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest
• All three reading frames (A, B, C versions) are provided to facilitate in-frame cloning with the C-terminal peptide
• α-factor secretion signal for directing secreted expression of the recombinant protein
• Zeocin resistance gene for selection in both E. coli and Pichia
• C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant fusion protein

BLOCK-iT™ Lentiviral RNAi Expression System (Invitrogen™)

The pLenti6/BLOCK-iT™-DEST expression vector provided in the BLOCK-iT™ Lentiviral RNAi Expression System can be used to efficiently introduce and stably express short hairpin RNA (shRNA) in vivo from a lentiviral vector. A novel cloning process places an ~50-bp DNA oligonucleotide immediately following a U6 pol III promoter into the BLOCK-iT™ U6 entry vector. The oligonucleotide is designed to express RNA that forms a stem-loop structure containing the sense and antisense regions of your target gene of interest. This shRNA is then recombined into the pLenti6/BLOCK-iT™-DEST vector. After viral production and transduction, the shRNA driven by the U6 promoter becomes stably integrated as an RNAi cassette. The shRNA generated avoids the hosts defense mechanism and will be effective at producing the RNAi gene knockdown response (Figure 1).

The pLenti6/BLOCK-iT™-DEST vector (Figure 2) offers:

attR sites for efficient recombination with the attL-flanked U6 Gateway® entry vector containing the RNAi cassette
• All of the required components for efficient lentiviral packaging and delivery of the shRNA of interest
• Blasticidin selection marker for fast, efficient selection of stable cell lines expressing the shRNA Using the BLOCK-iT Lentiviral RNAi Expression System, long-term analysis of gene blocking in both dividing and non-dividing mammalian cell types and animal models can be achieved.

The BLOCK-iT™ RNAi U6 Entry Vector Kit allows streamlined cloning of shRNA target sequences for testing in transient experiments. Selected RNAi expression cassettes are quickly and efficiently recombined from the BLOCK-iT™ RNAi U6 entry vector into the pLenti6/BLOCK-iT™-DEST vector via a standard Gateway® LR recombination reaction (Figure 3).

pEF6/V5-His TOPO™ TA Expression Kit (Invitrogen™)

The pEF6/V5-His TOPO® TA Expression Kit is designed for high-level mammalian expression from the powerful human EF-1 α promoter with the added convenience of one-step TOPO® Cloning. The pEF6/V5-His-TOPO® vector includes:

Enhancer/promoter elements from the human elongation factor 1α subunit (hEF-1α) for high-level expression in mammalian cells

• The Blasticidin resistance gene for rapid selection of stable mammalian cell lines
• A C-terminal V5 epitope for efficient detection with an Anti-V5 Antibody
• A C-terminal polyhistidine (6xHis) sequence for rapid purification with nickel-chelating resin and detection with an Anti-His(C-term)
• Antibody