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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.

pEXP3-DEST Vector Kit (Invitrogen™)

The Expressway™ Lumio™ Expression and Detection System takes advantage of the Lumio™ recognition sequence, a small, six amino acid sequence (Cys-Cys-Pro-Gly-Cys-Cys). The Lumio™ detection reagent binds the recognition sequence with high specificity and affinity, resulting in a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection. In addition, Expressway’s specialized E. coli lysate, derived from a slyD mutant, eliminates nonspecific binding of the Lumio™ Green Detection Reagent to the endogenous SlyD protein and provides optimal background for detection of recombinant proteins (Figure 1). The Lumio™ Green Detection Kit is included in the system for real-time detection of protein synthesis as well as easy in-gel protein detection. The Lumio™ vectors (Figures 2 and 3) also feature:

attR sites for efficient recombination with any attL-flanked Gateway® entry vector
N-terminal Lumio™ tag (pEXP3-DEST vector) with TEV cleavage site for efficient removal of the Lumio™ sequence following purification
C-terminal Lumio™ tag (pEXP4-DEST vector) for easy, and immediate in-gel detection
T7 promoter, ribosome binding site, and T7 terminator optimally spaced for cell-free protein expression

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]

GeneArt™ pYES1L Vector with Sapphire™ Technology (Invitrogen™)

GeneArt® pYES1L Vector with Sapphire Technology™ is a ready-to-use, 9.3 Kb linearized BAC⁄YAC shuttle vector, used for the assembly of DNA fragments with the GeneArt® High-Order Genetic Assembly System (cat#A13285 & A13286). The vector has the capacity to clone up to 110 Kb of DNA fragments. This product only includes the linearized GeneArt® pYES1L Vector with Sapphire Technology™.

Some of the key features and elements of the GeneArt® pYES1L Vector with Sapphire Technology™ include:
TRP1 to allow selection of yeast transformants in tryptophan-deficient medium
• ARS⁄CEN origin to allow stable maintenance of the construct in yeast
• F’ for conjugation
• oriT for maintenance of large constructs in E.coli
• Spectinomycin resistance for selection in E.coli
• 6 Convenient and validated restriction sites flanking the cloning region

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

The Thermo Scientific pTK-Gaussia Luc vector is a derivative of the pMCS-Gaussia Luc vector containing the Gaussia luciferase gene under the control of the 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 pCMV-Gaussia Luc vector:

• Weak (TK) constitutive promoters for co-transfection and normalization
• Naturally-secreting Gaussia luciferase gene, optimized for high expression 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

The Gaussia Luc Vectors contain a gene cloned from the copepod, Gaussia princeps. The gene encodes a naturally-secreted bioluminescent Gaussia luciferase (approx. 20kDa), which enables measurement of the reporter activity in media (for real-time assays) and in cell lysates.

These vectors are subject to a limited use label license.

More Product Data
Highly sensitive multiplex luciferase reporter assays
Luciferase assays in hard-to-transfect Jurkat cells
Monitoring neuronal differentiation using multiplexed luciferase reporters
Activation of the antioxidant response pathway by pesticide chemicals

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

pFLD1 Pichi pastoris Expression Vector Kit (Invitrogen™)

pFLD and pFLD-alpha are vectors used to express recombinant proteins in Pichia pastoris. Recombinant proteins are expressed as fusions to a C-terminal peptide containing the V5 epitope and a polyhistidine (6xHis) tag.

The vector allows high-level, methanol- and methylamine-inducible expression of the gene of interest in Pichia, and can be used in any Pichia strain, including X-33, GS115, SMD1168H, and KM71H. The pFLD vectors contain the following elements:

• 5' fragment containing the FLD1 promoter for methanol- or methylamineinduced expression of the gene of interest
• Zeocin™ resistance gene for selection in both E. coli and Pichia
• Ampicillin resistance gene for selection in E. coli
• C-terminal peptide containing the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant fusion protein (if desired)

pcDNA™4/TO Mammalian Expression Vector (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.

pPIC9K Pichia Vector (Invitrogen™)

The pPIC9K vector carries the kanamycin resistance gene that confers resistance to Geneticin® Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin® Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin® Reagent. The ability to grow in high concentrations of Geneticin® indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome. The pPIC9K vector directs secretion of expressed proteins. This vector is included in the Multi-Copy Pichia Expression Kit.

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

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

The pNF-κB Tluc16-DD Vector is a transcriptional reporter vector designed to monitor the activation of NFκB protein and NFκB-regulated signal transduction pathways in mammalian cells. The vector encodes the smallest known luciferase, TurboLuc™16 (Tluc16) luciferase, as the reporter under the control of a combination of an optimized minimal core promoter and 5 tandem repeats of the NFκB transcriptional response element.

• Intracellular Tluc16 luciferase gene, optimized for high expression in mammalian systems
• Dual-destabilization (DD) technology reduces accumulation of Tluc16 luciferase mRNA and protein in cells, enhancing the responsiveness of the assay
• Monitor activation of NF-κB protein and NF-κB-regulated signal transduction pathways in mammalian cells

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 pNF-κB Tluc16-DD luciferase expression vector also contains dual-destabilization (DD) technology that reduces accumulation of the 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 NFκB response elements to minimize non-specific transcriptional read-through. The Tluc16 luciferase activity can be measured using the Thermo Scientific TurboLuc™ One-Step Glow Assay Kit.

pTracer™-CMV/Bsd Mammalian Expression Vector (Invitrogen™)

Two Tracer™ mammalian expression vectors are available that express GFP fused to the selectable marker Blasticidin. Blasticidin is a potent selection agent that allows a more rapid generation of stable cell lines than other selection agents. Each vector uses a different set of promoters to express the gene of interest and the Cycle 3 GFP-Blasticidin resistance gene fusion.

The pTracer™-CMV/Bsd vector offers:

• CMV promoter for high-level constitutive expression of your gene of interest
• EF-1 promoter for high-level constitutive expression of the Cycle 3 GFP-Blasticidin resistance fusion

The pTracer™-EF/Bsd vectors offer:
• EF-1 promoter for high-level constitutive expression of your gene of interest
• CMV promoter for high-level constitutive expression of the Cycle 3 GFP-Blasticidin resistance fusion
• C-terminal V5 epitope tag for simple detection of recombinant protein 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
• Three reading frames for easier in-frame cloning

Gateway™ pDONR™/Zeo 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™/Zeo vector haa a pUC origin for high plasmid yields and universal M13 sequencing sites for ease of use.

pShooter™ Mammalian Expression Vector (pCMV/myc/ER) (Invitrogen™)

The pShooter™ vectors are designed to localize recombinant proteins to the nucleus, mitochondria, endoplasmic reticulum, or cytoplasm. In addition to the localization signal, each pShooter™ vector contains:

• Strong mammalian promoter (CMV or EF-1É) for constitutive expression of your protein of interest
• C-terminal c-myc epitope for detection with an Anti-myc Antibody
• Bovine Growth Hormone (BGH) polyadenylation site for mRNA stability
• f1 origin for single-stranded rescue of sense DNA
• Neomycin resistance gene for selection of mammalian cells with Geneticin¤ selection agent
• Ampicillin resistance and pUC origin for selection and maintenance in E. coli

Each vector is provided with a positive control plasmid. The positive control expresses GFP and targets it to the same subcellular location as the pShooter™ vector.

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™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).