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pJTI™ R4 CMV-TO MCS pA Vector

The pJTI™ R4 CMV-TO MCS pA vector is designed for the expression of your gene of interest under the control of the tet-inducible CMV promoter after restriction enzyme cloning and retargeting into the genomic R4 site of a Jump-In™ parental cell line.

This vector can be used for inducible or constitutive expression of your gene of interest, depending on which Jump-In™ parental cell line you use. When this vector is retargeted into a Jump-In™ T-REx™ parental cell line, gene expression is controlled by the tet-operon and can be induced by adding doxycycline to the growth media. When used with a Jump-In™ parental cell line such as the Jump-In™ GripTite™ HEK293 cell line, constitutive expression is achieved after retargeting. The R4 sites in the Jump-In™ parental cell lines result in a high retargeting efficiency, requiring less effort and less time than traditional cell engineering methods. Retargeting of Jump-In™ parental cell lines results in creation of an isogenic pool that is sufficient for cell-based experiments without the need for clonal selection. Alternatively, the high retargeting efficiency allows for easy selection of a positive stable clone for expressing your gene of interest.

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

The pcDNA™3.1/V5-His TOPO® TA Expression Kit offers one-step cloning of Taq-amplified PCR products into a high-level expression vector. Topoisomerase activation of the pcDNA3.1/V5-His-TOPO® vector allows PCR products to be ligated in just 5 minutes on your bench top and results in 90% recombinants. This kit is specifically formatted for storage in Invitrogen Supply Centers at -80°C. In addition, the vector includes the following features: Strong CMV promoter for high-level, constitutive expression V5 epitope tag for efficient detection of recombinant proteins 6×His sequence for purification using nickel-chelating resin and detection

Gateway™ pDEST™27 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/V5-His TOPO™ TA Expression Kit (Invitrogen™)

The pcDNA™3.1/V5-His TOPO® TA Expression Kit offers one-step cloning of Taq-amplified PCR products into a high-level expression vector. Topoisomerase activation of the pcDNA3.1/V5-His-TOPO® vector allows PCR products to be ligated in just 5 minutes on your bench top and results in 90% recombinants.

In addition, the vector includes the following features:

• Strong CMV promoter for high-level, constitutive expression.
• C-terminal V5 epitope tag for efficient detection of recombinant proteins with an Anti-V5 antibody.
• C-terminal polyhistidine (6xHis) sequence for purification using nickel-chelating resin and detection with an Anti-His (C-term) antibody.

pcDNA™4/HisMax TOPO™ TA Expression Kit (Invitrogen™)

pcDNA™4/HisMax is specifically designed to maximize protein expression in a variety of mammalian cells. The vector contains the QBI SP163 translational enhancer to increase expression levels two- to five-fold above those seen with the promoter alone (Figure 1). In addition to SP163-enhanced expression, pcDNA™4/HisMax includes a cleavable N-terminal Xpress™ tag for rapid detection of recombinant protein with an Anti-Xpress™ Antibody (Figure 2). pcDNA™4/HisMax is available TOPO® Cloning-ready for five-minute cloning of Taqamplified PCR products.

pcDNA™3.2/GW/D-TOPO™ Expression Kit (Invitrogen™)

The pcDNA™ vectors are designed for high-level, constitutive expression in a variety of mammalian cell lines. The pcDNA3.2/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
•Neomycin resistance gene for 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.

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.

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

The Thermo Scientific™ pMCS minP-Tluc16-DD Vector is a multiple cloning site (MCS) vector designed to accept a response element sequence lacking promoter activity for study of gene regulation using the intracellular TurboLuc™16 (Tluc16) luciferase reporter.

• Intracellular Tlac16 luciferase gene, optimized for high expression in mammalian systems
• Optimized minimal core promoter (minP) and 5′ UTR region for efficient expression of Tluc16 luciferase
• Multiple cloning sites provide versatility for transfer of regulatory elements from one vector to another
• Dual-destabilization (DD) technology reduces accumulation of Tluc16 luciferase mRNA and protein in cells, enhancing the responsiveness of the assay
• Synthetic polyA terminator and Transcriptional Pause Site (TPS) included upstream of MCS to minimize non-specific transcriptional read-through

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 expression vector also contains dual-destabilization (DD) technology that reduces accumulation of Tluc16 luciferase mRNA and protein in cells, enhancing the responsiveness of the assay. A synthetic polyA terminator and a Transcriptional Pause Site (TPS) are included upstream of the MCS to minimize non-specific transcriptional read-through. The Tluc16 luciferase activity can be measured using the Thermo Scientific TurboLuc™ One-Step Glow Assay Kit.

Gateway™ pEF-DEST51 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 forward-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

Vivid Colors™ pcDNA™6.2/C-EmGFP-GW/TOPO™ Mammalian Expression Vector (Invitrogen™)

The Vivid Colors™ pcDNA™6.2 Fluorescent Protein TOPO® Expression Vectors (Figure 1) allow you to rapidly clone your gene and fuse it to the widely used and well characterized Fluorescent Proteins (FPs) from the jellyfish Aequorea victoria (1, 2). These powerful TOPO® cloning vectors contain the Emerald Green Fluorescent Protein (EmGFP) or the Yellow Fluorescent Protein (YFP) for simple, non-invasive detection of recombinant protein (Figure 2). Both FPs have been humanized for optimal mammalian expression (3). The Vivid Colors™ pcDNA™6.2 Fluorescent Protein TOPO® Expression Vectors offer:
• Topoisomerase I for one-step, 5-minute TOPO® cloning of your PCR-amplified gene of interest
• CMV promoter for high-level expression of the recombinant fluorescent fusion protein
• Ability to fuse EmGFP or YFP to the N- or C-terminus of your protein
• Bsd resistance marker for rapid selection of stable cell lines

pBudCE4.1 Mammalian Expression Vector (Invitrogen™)

The pBudCE4.1 vector is designed for the independent expression of two genes from a single plasmid in mammalian cells. Using pBudCE4.1 to generate stable mammalian expression cell lines ensures that there is an equivalent copy number of each gene in the cell. This can eliminate variable expression due to differences in gene copy number. pBudCE4.1 provides expression cassettes withthe following features:

The CMV promoter for high-level transcription of genes with an optional c-myc epitope tag for rapid detection and 6xHis sequence for simple purification

• The human EF-1α promoter for high-level expression of genes with an optional V5 epitope tag for rapid detection and 6xHis sequence for simple purification
• The Sh ble (ZeoR) gene for efficient selection in both mammalian cells and E. coliwith the selection agent Zeocin™

GeneSwitch™ Mammalian Expression Kit, core kit (Invitrogen™)

Switch-On Expression from the Lowest Basal Levels
The GeneSwitch™ System for inducible mammalian expression is ideal for experiments that require the absolute lowest uninduced expression levels. The expression vector pGene/V5-His provides a minimal promoter, GAL4-E1b, consisting of the binding sites for the yeast Gal4 DNA binding protein followed by the TATA sequence from the Adenovirus E1b promoter. Without additional factors, the GAL4-E1b promoter is transcriptionally silent.

To activate transcription from the GAL4-E1b promoter, the GeneSwitch™ regulatory protein is expressed from a minimal TK promoter on the pSwitch vector. The GeneSwitch™ protein has three functional domains:

• Gal4 DNA Binding Domain (Gal4-DBD) to bind the regulatory protein to the GAL4-E1b promoter
• Truncated human Progesterone Receptor Ligand Binding Domain (hPR-LBD) that undergoes a conformational change when it binds the progesterone antagonist, mifepristone
• Transcription activation domain from the NFØB transcription factor p65 (p65AD) to activate transcription from the silent GAL4-E1b promoter

In the absence of mifepristone, the conformation of the hPR-LBD region prevents the GeneSwitch™ regulatory protein from activating transcription from the GAL4-E1b promoter. When mifepristone is added and binds the hPR-LBD region, the GeneSwitch™ regulatory protein assumes a conformation that permits it to stimulate transcription from the GAL4-E1b promoter (Figures 1 and 2).

Induction of the GeneSwitch™ System leads to activation of the gene of interest on the vector pGene/V5-His. In addition, four Gal4 binding sites upstream of the minimal HSV TK promoter on pSwitch can bind the pSwitch regulatory protein. Therefore, adding mifepristone up-regulates production of the regulatory protein (Figure 1). The increased levels of the GeneSwitch™ regulatory protein result in induction of the gene of interest from pGene/V5-His to levels that can approach those of viral promoters.

The GeneSwitch™ Kit offers exceptionally low uninduced and high induced expression levels in mammalian cells. The system provides the expression vector pGene/V5-His with the minimal, synthetic GAL4-E1b promoter that is transcriptionally silent until activated. The GeneSwitch™ regulatory protein binds the GAL4-E1b promoter to activate transcription upon the addition of mifepristone. In addition, the GeneSwitch™ protein upregulates its own expression, leading to amplified expression of the gene of interest from pGene/V5-His. pGene/V5-His offers several features that facilitate expression analysis and purification of recombinant proteins in mammalian cells:

• C-terminal V5 epitope tag for detection with Anti-V5 Antibodies
• C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with Anti-His(C-term) Antibodies
• The Zeocin™ resistance gene for effective selection in both mammalian cells and E. coli

ViraPower™ II Lentiviral N-Lumio™ Gateway™ Expression System (Invitrogen™)

To efficiently deliver and monitor expression of your gene in live cells, choose a Lentivirus expression vector to fuse your protein with Lumio™ and V5 epitope tag. Lumio™ technology is designed for simple fluorescent detection of recombinant protein. The Lumio™ tag is a six amino acid sequence that binds a fluorescent substrate, allowing you to visually detect your protein of interest in mammalian cells. Choose from either the C-terminal or N-terminal fusion tagged vectors for optimal expression. These Lentiviral Lumio™ Reporter vectors are configured for making high-titer Lentiviral virus preps for efficient viral delivery to both dividing and non-dividing cells and selection with blasticidin.The pLenti6.2/C,N-Lumio™/V5-DEST vectors provide:

• CMV promoter for high-level expression of your gene
• PGK promoter for long-term stable expression of the Blasticidin (bsd)-resistance marker
• Lumio™ tag for accurate in vivo and in vitro protein detection
• Sites for rapid recombinational cloning using Gateway® Technology