An Entry clone contains your gene of interest flanked by attL sequences, which are then used to recombine with attR sequences to create your desired expression clone. There are three methods you can use to produce an Entry clone: BP cloning, restriction enzyme and ligase cloning, and Invitrogen TOPO cloning into a Invitrogen Gateway Entry vector, which is the most common method.
Gateway Entry options
- RNAi expression—Efficient delivery and long-term stable or transient shRNA expression in any mammalian cell types with Lentiviral and Adenoviral Gateway vectors
- Viral expression—High level, stable and transient gene expression in any mammalian cell type
- In Vitro protein synthesis—Directly synthesize high yields of recombinant protein in a single reaction tube in just 2 hours without special equipment. Scale the reaction to achieve microgram to milligram levels of protein
- in vivo biotinylation—Easy efficient method for expressing, purifying and detecting biotinylated recombinant proteins.
- Tag-On-Demand technology—Produce native or tagged proteins from one vector.
The three possible methods that lead to the Entry clone are depicted in Figure 1. Using TOPO vectors and PCR amplification/restriction-enzyme vectors are the most common ways to construct your own Entry clone.
Figure 1. Strategies to build the Entry clone. The three possible methods that lead to the Entry clone are depicted: (A) BP cloning, (B) TOPO cloning, and (C) restriction enzyme and ligase cloning. Red arrows represent the fragment of interest. Adapted from Katzen F (2007) Expert Opin Drug Discov 2(4):571–589.
TOPO cloning
We offer two types of TOPO vectors that offer easy TOPO cloning to create a Gateway Entry vector. pCR8/GW/TOPO vector kits and pENTR/D-TOPO vector families both offer 5-minute, efficient TOPO cloning, with >95% efficiency.
pENTR/D-TOPO Vector kits
pENTR/D-TOPO vectors take advantage of fast, efficient Directional TOPO cloning that delivers your insert in the correct orientation for expression. These vectors contain the necessary attL sequences for recombination into any Destination vector and certain versions carry a TEV protease cleavage site for producing native proteins after expression (Figure 2).
Figure 2.Several pENTR vectors are available for Directional TOPO cloning and direct access to the multitude of Gateway expression vectors.
pCR8/GW/TOPO Vector kits
The pCR8/GW/TOPO vector enables efficient TOPO-TA cloning. These vectors easily facilitate multipurpose use: rapid recombination into a variety of Gateway Destination vectors, convenient sequencing, robust selection in E. coli with spectinomycin resistance, and easy excision of insert DNA with flanking EcoR I sites (Figure 3).
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Figure 3.The pCR8/GW/TOPO Entry vector allows TOPO TA Cloning for multiple downstream applications. |
| Rating | Kit | Cat. No. | Advantages |
|---|---|---|---|
| Vectors for TOPO cloning | |||
| BEST High efficiency, directional TOPO Cloning Vector Kits | pENTR/TEV/D-TOPO Cloning Kit | K253520 |
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| pENTR/D-TOPO Cloning Kit | K240020 |
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| pENTR/SD/D-TOPO Cloning Kit | K242020 |
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| pENTR/TEV/D-TOPO Cloning Kit | K252520 |
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| VERY GOOD High efficiency TOPO TA Cloning Vector Kits | pCR8/GW/TOPO TA Cloning kit | K252002 |
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| pCR8/GW/TOPO TA cloning kit | K252020 |
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| pCR8/GW/TOPO TA Cloning Kit | K250020 |
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| Vectors for PC amplification or restriction enzyme cloning | |||
| Standard restriction cloning Vectors | pENTR 1A Dual Selection Vector | A10462 |
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| pENTR 2B Dual Selection Vector | A10463 |
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| pENTR 3C Dual Selection Vector | A10464 |
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| pENTR 4 Dual Selection Vector | A10465 |
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| pENTR 11 Dual Selection Vector | A10467 |
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Purchasing a premade clone
You can also utilize Gateway technology with a ready-to-use clone from our extensive clone collection. The Thermo Scientific™ Ultimate™ ORF Clone Collection consists of high-quality, full-insert sequenced human and mouse open reading frames already cloned into the pENTR 221 Gateway Entry vector for limitless downstream analysis capabilities. Clones contain DNA- and amino acid sequence–verified, expression-ready cDNAs, including kinases, G-protein–related, phosphatases, ion channels, GPCRs, chemokines, nuclear receptors, and cytokines.