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pMT/BiP/V5-His A, B, & C Drosophila Expression Vectors (Invitrogen™)

The DES®-Inducible/Secreted Kit provides the vector pMT/BiP/V5-His for inducible, secreted expression of recombinant proteins. This vector offers the inducible metallothionein promoter that is induced upon addition of copper sulfate or cadmium chloride. The N-terminal signal sequence from the insect BiP gene is provided to direct the recombinant fusion protein through the secretory pathway of S2 cells into the culture medium. The pMT/BiP/V5-His vector offers the following additional features:

• Small size (3.6 kb) to improve DNA yields and increase subcloning efficiency
• C-terminal V5 epitope tag for rapid detection with Invitrogens Anti-V5 Antibody
• C-terminal 6xHis tag for simple purification of recombinant fusion proteins using nickel-chelating resin

To facilitate cloning, a set of three vectors-A, B, and C-is provided. Each vector has the multiple cloning site in a different reading frame relative to the BiP signal sequence.

pEF1/myc-His A, B, & C Mammalian Expression Vectors (Invitrogen™)

All pEF or pUB 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.

pGAPZ A, B, & C Pichia pastoris Expression Vectors (Invitrogen™)

pGAPZ A, B, & C and pGAPZ A, B, & C are expression vectors designed for high-level, constitutive expression in Pichia pastoris. pGAPZ and pGAPZ were created by replacing the methanol-regulated AOX1 promoter with the constitutive, glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in the backbone of the pPICZ vectors. Although the yield
of any protein constitutively expressed in the Pichia system is dependent on the toxicity of the protein to yeast, constitutive expression under the control of pGAPZ or pGAPZ vectors can sometimes produce greater yields than inducible expression (1).

pcDNA™3.1/V5-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.

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

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

The pPICZ A, B, & C Pichia Vectors are designed for simple cloning and selection, high-level expression, and rapid detection and purification of the recombinant protein. These vectors contain the Zeocin™ resistance gene for direct selection of multi-copy integrant strains. By selecting with increasing amounts of Zeocin™, strains with multiple copies of your gene of interest integrated into the genome are obtained. Increasing the number of copies of the gene of interest in a recombinant Pichia strain can result in higher expression levels. The vectors are included in the EasySelect™ Pichia Expression Kit (Cat. No. K1740-01).

Features of the pPICZ vectors include:

• Inducible AOX1 promoter for high-level expression in Pichia pastoris
c-
myc epitope tag for convenient detection with an Anti-myc Antibody
• C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with an Anti-His(C-term) Antibody
• Zeocin™ resistance for direct selection of multi-copy integrants

Episomal iPSC Reprogramming Vectors (Invitrogen™)

Episomal iPSC Reprogramming Vectors are a cost-effective mixture of three vectors designed to provide the optimal system for generating transgene-free and virus-free induced pluripotent stem cells (iPSCs) in a feeder-free environment. Originally developed by Junying Yu and James Thomson(1) and further optimized by Cellular Dynamics International, these Episomal iPSC Reprogramming Vectors have proven successful in reprogramming a number of different somatic cell types.
• Safe for all stages of your iPSC research—transgene-free and viral-free reprogramming allows use from basic to pre-clinical research
• Reprogram a variety of somatic cell types—provides flexibility in somatic cell selection
• Optimized for feeder-free reprogramming—allows for defined and feeder-free reprogramming when used with Essential 8™ Medium

Create Transgene- and Virus-free iPSCs
The Episomal iPSC Reprogramming Vectors are a well-described system for producing transgene-free, virus-free iPSCs, providing a source of iPSCs for all stages of your pluripotent stem cell research. Other reprogramming methods, such as lentivirus, contain transgenes that can integrate into the host genome, potentially disrupting the genome or causing unpredictable results. These episomal vectors are introduced into the cell by electroporation. As oriP/EBNA1 vectors, they contain all 6 reprogramming factors (Oct4, Sox2, Nanog, Lin28, Klf4 and lMyc) and replicate extrachromosomally only once per cell cycle. At this replication rate, the episomes are lost at a rate of approximately 5% per cell generation.

Generate iPSCs from a Wide Variety of Somatic Cell Types
iPSCs have been generated with episomal vectors from a range of somatic cells including fibroblasts, bone marrow mononuclear cells, PBMCs, lymphoblast B cells, and various disease-type fibroblasts and PBMCs. Each kit provides enough material for 6 reprogramming experiments.

Optimized for Feeder-free Reprogramming with Essential 8™ Medium
The Episomal iPSC Reprogramming Vectors were designed in the laboratories of James Thomson and Cellular Dynamics International for use with Essential 8™ Medium, thus providing an optimal environment for defined, feeder-free reprogramming.

Commercialized in Partnership with Cellular Dynamics International.

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

Reference:
1. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences; Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson J. Science. 2009; 324:797-801.

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

The Vivid Colors™ Fluorescent Protein Gateway® Destination Vectors allow you to quickly and easily fuse a protein of interest to the widely used and well-characterized fluorescent proteins from the jellyfish Aequorea victoria (1,2) using the pcDNA™ 6.2 Gateway™ mammalian expression vector. These powerful Gateway® Technology vectors contain the next-generation EGFP, Emerald Green Fluorescent Protein (EmGFP), or the popular Yellow Fluorescent Protein (YFP) for simple, non-invasive detection of recombinant protein. Both fluorescent proteins have been humanized for optimal mammalian expression (3). In addition, the Vivid Colors™ pcDNA™ 6.2 Fluorescent Protein Gateway® Vectors offer:

• CMV promoter for high-level expression of the recombinant fluorescent fusion protein
• Options to fuse EmGFP or YFP to the N- and C-terminus of your protein

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

The Thermo Scientific pCMV-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 strong Cytomegalovirus (CMV) 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-Dura Luc vector:

• Strong (CMV) 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
pTK-Gaussia-Dura Luc Vector for Luciferase Assays

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

The Vivid Colors™ Fluorescent Protein Gateway® Destination Vectors allow you to quickly and easily fuse a protein of interest to the widely used and well-characterized fluorescent proteins from the jellyfish Aequorea victoria (1,2) using the pcDNA™ 6.2 Gateway™ mammalian expression vector. These powerful Gateway® Technology vectors contain the next-generation EGFP, Emerald Green Fluorescent Protein (EmGFP), or the popular Yellow Fluorescent Protein (YFP) for simple, non-invasive detection of recombinant protein. Both fluorescent proteins have been humanized for optimal mammalian expression (3). In addition, the Vivid Colors™ pcDNA™ 6.2 Fluorescent Protein Gateway® Vectors offer:

• CMV promoter for high-level expression of the recombinant fluorescent fusion protein
• Options to fuse EmGFP or YFP to the N- and C-terminus of your protein

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

Champion™ pET302/NT-His and pET303/CT-His Vector Kit (Invitrogen™)

The Champion™ pET302/NT-His and pET303/CT-His Vector Kit is designed for cloning a gene of interest via restriction enzyme(s) and ligase (REaL) and subsequent high-level expression from 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 (pET302/NT-His and pET303/CT-His, respectively) for convenient purification and detection (Figure 1). Expression levels obtained from these vectors may be higher than those obtained from another suppliers pET vector (Figure 2). To maximize expression, use with MagicMedia™ E. coli Expression Medium.


Contents and Storage:

The Champion™ pET302/NT-His and pET303/CT-His Vector Kit includes 6 µg each of pET302/NT-His and pET303/CT-His and 10 µg of a control vector. Store at -20“C. All components are guaranteed stable for 6 months when properly stored.

Vivid Colors™ pcDNA™6.2/C-YFP-DEST Vector (Invitrogen™)

The Vivid Colors™ Fluorescent Protein Gateway® Destination Vectors allow you to quickly and easily fuse a protein of interest to the widely used and well-characterized fluorescent proteins from the jellyfish Aequorea victoria (1,2) using the pcDNA™ 6.2 Gateway™ mammalian expression vector. These powerful Gateway® Technology vectors contain the next-generation EGFP, Emerald Green Fluorescent Protein (EmGFP), or the popular Yellow Fluorescent Protein (YFP) for simple, non-invasive detection of recombinant protein. Both fluorescent proteins have been humanized for optimal mammalian expression (3). In addition, the Vivid Colors™ pcDNA™ 6.2 Fluorescent Protein Gateway® Vectors offer:

• CMV promoter for high-level expression of the recombinant fluorescent fusion protein
• Options to fuse EmGFP or YFP to the N- and C-terminus of your protein

T-REx™ Complete Kit, with pcDNA™4/TO© 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 provided through two tetracycline operator sequences (TetO2) that 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. 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.

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.

Vivid Colors™ pcDNA™6.2/N-YFP-DEST Vector (Invitrogen™)

The Vivid Colors™ Fluorescent Protein Gateway® Destination Vectors allow you to quickly and easily fuse a protein of interest to the widely used and well-characterized fluorescent proteins from the jellyfish Aequorea victoria (1,2) using the pcDNA™ 6.2 Gateway™ mammalian expression vector. These powerful Gateway® Technology vectors contain the next-generation EGFP, Emerald Green Fluorescent Protein (EmGFP), or the popular Yellow Fluorescent Protein (YFP) for simple, non-invasive detection of recombinant protein. Both fluorescent proteins have been humanized for optimal mammalian expression (3). In addition, the Vivid Colors™ pcDNA™ 6.2 Fluorescent Protein Gateway® Vectors offer:

• CMV promoter for high-level expression of the recombinant fluorescent fusion protein
• Options to fuse EmGFP or YFP to the N- and C-terminus of your protein

T-REx™ Core Kit, with pcDNA™4/TO 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.

Gateway™ pcDNA™-DEST53 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.

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)

pLenti6/TR Vector Kit (Invitrogen™)

The pLenti6/TR lentiviral vector can be used to make stable cell lines expressing high levels of tetracycline repressor (TR) protein under the control of the CMV promoter. This vector contains elements that allow packaging of the construct into lentiviral particles and the Blasticidin resistance marker for selection of stable cell lines (Figure 1). The pLenti6/TR vector is designed for use with the BLOCK-iT™ Inducible H1 Lentiviral RNAi System, BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit, and the ViraPower™ T-REx™ Lentiviral Expression System.

pJTI™ R4 Dest CMV N-EmGFP pA Vector

The pJTI™ R4 Dest CMV N-EmGFP pA vector allows a gene of interest to be expressed as an N-terminal Emerald Green Fluorescent Protein (EmGFP) fusion in a Jump-In™ parental cell line. When used in combination with a Jump-In™ parental cell line kit such as the Jump-In™ GripTite™ HEK293 cell line (A14150), an isogeneic stable cell line can be created with less effort and in less time than traditional cell engineering methods. In addition, Gateway® technology increases the efficiency of the cloning steps to introduce your GOI into the Gateway®-ready pJTI™ R4 Dest CMV N-EmGFP pA vector. The high retargeting efficiency, made possible by the R4 sites in the Jump-In™ parental cell line, allows the use of the isogenic pool for additional 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 with an N-terminal EmGFP.

The Jump-In™ Vectors and Parental Cell Lines Let You:

• Quickly and efficiently develop stably engineered isogenic cell pools in about half the time compared to traditional cell engineering methods.
• Utilize isogenic expression from a defined genomic locus as the ideal solution for comparative analysis of gene families, isoforms or orthologs.
• Generate multiple cell lines in parallel due to the simplified work flow.
• Easily access the Jump-In™ technology with the ability to generate an unlimited number of cell lines without complicated licenses or restrictions to interpret.

Save Time with Rapid and Efficient Generation of Engineered Cell Lines
Using Gateway® technology, you can generate an expression construct for retargeting using the pJTI R4 DEST CMV N-EmGFP pA destination vector. In combination with a Jump-In™ parental cell line kit, the expression cassette is efficiently and specifically inserted into a genomic target R4 site resulting in the generation of functional cell pools in as little as 2 weeks without laborious clone isolation and analysis. Even generation of clonal cell lines can be done with reduced time and effort due to the high percentage of positive clones.

Expand Your Experimental Capabilities
The pJTI™ R4 Dest CMV N-EmGFP pA vector along with a Jump-In™ parental cell line provide the ideal solution for cells and assays where transient engineering technologies are problematic, as well as for difficult to "engineer" cell lines. The kit also provides a convenient way to create target panels of gene families, isoforms, or orthologs. Genes coding for large proteins or multi-unit proteins are not a problem since the Gateway® destination vectors accept large inserts.

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

pVAX1 Vector (Invitrogen™)

pVAX1 is specifically designed for use in the development of DNA vaccines. pVAX1 has the following features:

• Eukaryotic DNA sequences limited to those required for expression in order to minimize the possibility of chromosomal integration
• Kanamycin resistance gene for selection in E. colibecause ampicillin has been reported to cause an allergic response in some individuals
• Expression levels of recombinant proteins from pVAX1 comparable to those achieved with its parent vector, pcDNA™3.1

The small size of pVAX1 and the variety of unique cloning sites simplify subcloning of even very large DNA fragments.

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.

BLOCK-iT™ Lentiviral RNAi Zeo Gateway™ Vector Kit (Invitrogen™)

The BLOCK-iT™ Lentiviral RNAi Zeo Gateway® Vector Kit contains the pLenti4/BLOCK-iT™-DEST expression vector which enables lentiviral delivery and genomic integration of DNA coding for shRNA. Once expressed, the shRNA is processed by cellular machinery and initiates target-specific RNAi. The pLenti4/BLOCK-iT™-DEST vector offers:

Gateway® Technology for efficient recombination of the RNAi cassette from the BLOCK-iT™ inducible pENTR™/H1/TO vector

• All required components for efficient lentiviral packaging, delivery, and integration of the shRNA
• Zeocin™ selection marker for fast selection of clonal cell lines containing the RNAi cassette

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.

pMCS Tluc16 Vector for Luciferase Assays (Thermo Scientific™)

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

• Intracellular Tluc16 luciferase gene, optimized for high expression in mammalian systems
• Multiple cloning sites provide versatility for transfer of regulatory elements from one vector to another
• 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. 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.

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]

pJTI™ R4 Exp CMV EmGFP pA Vector

The pJTI™ R4 Exp CMV EmGFP pA vector is a positive control vector for assessing the success of a retargeting reaction with a Jump-In™ parental cell line. When co-transfected with the integrase vector (pJTI™ R4 Int vector) included in the Jump-In™ parental kits, the EmGFP will be expressed and positive cells will fluoresce green.

Ensure the Success of Your Jump-In™ Retargeting Reactions
Successful retargeting of Jump-In™ parental cell lines like the the Jump-In™ GripTite™ HEK293 kit (A14150) 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 retargeting reaction with the pJTI™ R4 Exp CMV 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.

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

pT7CFE1-CGFP-HA-His Vector for Mammalian Cell-Free Protein Expression (Thermo Scientific™)

Thermo Scientific pT7CFE1-CGFP-HA-His 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-CGST-HA-His Vector is available with tandem affinity tags, GFP,HA and 6xHis at the C-terminus, to facilitate protein purification and detection. pT7CFE1-CGST-HA-His also has a cleavable tag, HRV 3C, available on the C-terminus.

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.

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Choosing a vector and purification method for in vitro protein expression

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BLOCK-iT™ Lentiviral RNAi Gateway™ Vector Kit (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).