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

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.

pcDNA™3.1/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.

pUC57 DNA Thermo Scientific™

Thermo Scientific Plasmid pUC57, 2710 bp in length, is a derivative of pUC19. pUC57 MCS contains 6 restriction sites with protruding 3’-ends that are resistant to E. coli exonuclease III. This vector is designed for cloning and generation of ExoIII deletions. The exact positions of the genetic elements are shown on the map (termination codons included). DNA replication initiates at position 890 (±1) and proceeds in the direction indicated. The bla gene nucleotides 2510-2442 (compl. strand) code for a signal peptide.

Highlights

• More than 90% in the supercoiled form
• Isolated from E. coli (dam+, dcm+)
• Purified by chromatography using proprietary patented technology

Applications

• Cloning
• Sequencing of insert DNA

Related Products
pUC18 DNA
pUC19 DNA

ViraPower™ UbC Lentiviral Gateway™ Expression Kit Invitrogen™

The ViraPower™ UbC Lentiviral Gateway® Expression Kit includes all the components needed to generate lentivirus, including vector kit, 293FT cell line, and the support kit. This kit 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⁄UbC⁄V5-DEST™ vector has the UbC promoter for driving constitutive but physiological levels of expression of the target gene and the blasticidin selection marker for stable selection in mammalian cells.

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

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

Kit includes
• pLenti6⁄UbC⁄V5-DEST™ Gateway® Vector (V499-10)
• ViraPower™ Bsd Lentiviral Support Kit (K4970-00)
• 293FT Cell Line (R700-07)

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

aLICator LIC Cloning and Expression Kit 4 (N-terminal His-tag/WQ) 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 Set 2 (All-in-One/WQ)
CloneJET PCR Cloning Kit
Fast DNA End Repair Kit
aLICator LIC Cloning and Expression Kit 1 (untagged)

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.

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

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pT7CFE1-NHA-CHis Vector for Mammalian Cell-Free Protein Expression
pT7CFE1-CGST-HA-His Vector for Mammalian Cell-Free Protein Expression
pT7CFE1-NHis-GST Vector for Mammalian Cell-Free Protein Expression
pT7CFE1-NHis-GST-CHA Vector for Mammalian Cell-Free Protein Expression

pTracer™-SV40 Mammalian Expression Kit Invitrogen™

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

All three pTracer™ vectors have the following features:

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

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

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

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

pMCS Tluc16-DD Vector for Luciferase Assays Thermo Scientific™

The Thermo Scientific™ pMCS Tluc16-DD 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 unique 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 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 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.

pCRE Tluc16-DD Vector for Luciferase Assays Thermo Scientific™

The pCRE Tluc16-DD Vector is a transcriptional reporter vector designed to monitor the activation of cAMP-binding protein (CREB) and cAMP-mediated 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 c-AMP response element (CRE).

• 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 the activation of cAMP-binding protein (CREB) and cAMP-mediated 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 pCRE 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 cAMP 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.

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.

pENTR™/SD/D-TOPO™ Cloning Kit, with One Shot™ Mach1™-T1R Chemically Competent E. coli Invitrogen™

The pENTR™/SD/D-TOPO® Cloning Kits utilize a highly efficient, 5-minute cloning strategy ("TOPO® Cloning") to directionally clone a blunt-end PCR product into a vector for entry into the Gateway® System or the MultiSite Gateway® System. Blunt-end PCR products clone directionally into the pENTR™/SD/D-TOPO® entry vector at greater than 90% efficiency, and include an upstream Shine-Dalgarno sequence for expression in prokaryotes.

The kit comes with everything necessary to clone and select your PCR amplified gene of interest:

Gateway® System Ready - Rapidly shuttle cloned genes between multiple vector systems
Fast and Easy - Go from PCR to Gateway® Entry clone in just 3 steps and as little as ~5 minutes hands on time
Efficient - Achieve over 90% clones with correct insert in the right direction
Proven - Reliable performance for over a decade

pENTR™/SD/SD D-TOPO® Cloning Kits Overview

VECTOR

pENTR™ /SD SD/SD D-TOPO® Vector Directional cloning vector for entry to the Gateway® System and optimized for prokaryotic expression

CLONING METHOD

Directional TOPO® Cloning Topoisomerase I based ~5 minute directional ligation of blunt-end proofreading polymerase-amplified PCR products to the vector

COMPETENT CELLS

Two Options Choose from kits with either high efficiency, or fast growing competent cells
Simple Access to the Gateway® System
For access to the Gateway® System, just PCR amplify your gene of interest and add the product straight to the provided topoisomerase charged pENTR™ /SD SD/SD D-TOPO® vector, incubate 5 minutes, and transform the provided competent E. coli cells. The resulting attL containing Gateway® Entry clones are ready for efficient recombination with your choice of Gateway® Destination vectors.

Optimized pENTR™ /SD SD/SD D-TOPO® Vector
The pENTR™ /SD SD/SD D-TOPO® vector (Figure 1) includes a T7 gene 10 translational enhancer and a ribosome binding site (RBS) for optimal expression of native protein after recombination with a prokaryotic Gateway® destination vectors. You can also use the vector for expression in other hosts systems by recombination with different destination vectors. The vector has M13 and T7 primer sequencing sites and attL recombination sites flanking the PCR product insertion site. This allows clones to be easily sequence verified and recombined into your choice of attR containing Gateway® destination vectors. A Kanamycin resistance gene and a pUC origin are used for selection and high-copy propagation in E. coli.

Simplified Directional Cloning
With Directional TOPO® cloning technology there is no need for PCR clean up, vector preparation, or other time-intensive DNA manipulation steps. Just add your PCR reaction straight to the provided topoisomerase charged vector, incubate 5 minutes, transform, and obtain up to 90% directionally inserted clones. A four base overhang on the vector pairs with a four base sequence designed into the forward primer used in your PCR reaction to provide directionality to the topoisomerase ligation reaction (Figure 2).

The Power of Gateway® Recombination Cloning Technology
Gateway® recombination cloning technology circumvents the limitations of restriction mediated cloning, enabling you to access virtually any expression system in a simple one hour, 99%-efficient and reversible, Gateway® recombination reaction. The ability to move the same sequence of DNA between different vectors without using restriction enzymes, ligase, subcloning steps, screening of countless colonies, or re-sequencing will help save you time, money, and effort.

Leading Cloning Technologies
When it comes to cloning, TOPO® cloning technology and Gateway® recombination cloning technology have been a reliable partner for thousands of scientists for over ten years. Fast, simple to use, and efficient, TOPO® cloning and Gateway® recombination allow for rapid cloning and subsequent transferring of genes between a wide assortment of Gateway® expression vectors.

Kit Options
The pENTR™/SD/D-TOPO® Cloning Kit can be purchased with either TOP10 competent cells for standard cloning or Mach1™-T1R competent cells for fast growth.

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

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

TOPO™ TA Cloning™ Kit for Sequencing, with pCR™4-TOPO™ Vector, One Shot™ TOP10 Chemically Competent E. coli, and PureLink™ Quick Plasmid Miniprep Kit Invitrogen™

The TOPO® TA Cloning® Kits for Sequencing provide a highly efficient, 5 minute, one-step cloning strategy ('TOPO® Cloning') for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector for sequencing. Each kit uses the pCR™4-TOPO® TA vector with specially designed sequencing primer sites that return more insert sequence and less vector sequence from each reaction. These kits include everything necessary to clone and select recombinant vectors containing your PCR fragment of choice.

Get More Sequence—Allows for more insert sequence and less vector seuquence when using standard sequencing primers
Fast and Easy—Go from PCR-to-clones in just 3 steps and in as little as 5 minutes hands-on time
Efficient—Achieve up to 95% clones with correct insert
Proven—Reliable performance for over a decade with over 4000 citations

TOPO® TA Cloning® Kits for Sequencing Kit Overview
VECTOR: pCR™4-TOPO® TA Vector—Optimized cloning vector for improved sequencing results
CLONING METHOD: TOPO® TA Cloning —Topoisomerase I based 5 minute ligation of PCR products with 3´A overhangs (Taq-amplified) to the vector
COMPETENT CELLS: Various Options —Choose from kits with either general, high-efficiency, bacteriophage T1-resistant, or fast-growing competent cells

pCR™4-TOPO® TA VectorOptimized for Sequencing
We have removed much of the multiple cloning site from the pCR™4-TOPO® TA vector to shorten the distance between sequencing primer sites and the insert site to as little as 33 bp. This means sequencing reactions give less vector sequence and more insert sequence. The pCR™4-TOPO® TA vector has sites for 4 common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.

pCR™4-TOPO® TA Clone Selection and Manipulation
The pCR™4-TOPO® TA vector contains both ampicillin and kanamycin resistance markers and a LacZα-ccdB gene fusion for positive selection and blue/white screening. The vector’s minimized multiple cloning site still includes flanking EcoRI sites for simplified excision of cloned PCR products and a unique Sse8387I site for generation of nested deletions prior to sequencing. T7 and T3 promoters are also present for in vitro transcription.

Simplified TOPO®-Based Cloning
Using TOPO® cloning technology, there is no need for PCR primers containing specific sequences, post-PCR procedures, vector preparation, or other time-intensive DNA manipulation steps. Just add your PCR reaction straight to the provided topoisomerase-charged vector, incubate 5 minutes, and transform with the providedE. coli competent cells.

Efficient Cloning
With up to 95% of clones carrying the desired insert, you can screen less clones to help you save time and money. The pCR™4-TOPO® TA vector used in this kit comes with 3´T overhangs for efficient ligation of Taq-amplified PCR products with 3´A overhangs.

The Standard in Cloning
When it comes to cloning, TOPO® cloning technology has been a reliable partner for thousands of scientists for over ten years. Fast, simple-to-use, and efficient, TOPO® cloning has been applied to many different vectors for a wide array of applications.

TOPO® TA Cloning® Kits for Sequencing Kit Options
The TOPO® TA Cloning® Kit for Sequencing can be purchased with a variety of competent cells that deliver different advantages depending on what your needs are:

• General cloning: TOP10 (Cat. No. K4575-J10, K4575-01, K4575-40)
• High efficiency cloning: TOP10 Electrocomp™ Cells (Cat. No. K4580-01, K4580-40)
• General cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. No. K4595-01, K4595-40)
• Fast growth: Mach1™-T1R Chemically Competent E. Coli (Cat. No. K4530-20)

We also offer a version of the kit that includes a PureLink™ Quick Plasmid Miniprep Kit (Cat. No. K4575-02) for use in isolation of clean, sequencing-ready, recombinant plasmid.

TOPO® products are For Research Use Only. Not intended for any animal or human therapeutic of diagnostic use.

Related Links
Custom Vector Construction and Cloning Services
Plasmid DNA Purification Kit Selection Guide
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pT7CFE1-NHis-GST Vector for Mammalian Cell-Free Protein Expression Thermo Scientific™

Thermo Scientific pT7CFE1-NHis-GST 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-NHis-GST Vector is available with tandem affinity tags, 9xHis and GST at the N-terminus, to facilitate protein purification and detection. pT7CFE1-NHis-GST also has a cleavable tag, HRV 3C, available on the N-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.

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

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