Gateway® Entry Clones
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 TOPO® cloning into a 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 below: 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.
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 1).
Figure 1. Several pENTR™ vectors are available for Directional TOPO® cloning and direct access to the multitude of Gateway® expression vectors.
pCR®8/GW/TOPO® Vector Kits
The pCR®8/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 exicision of insert DNA with flanking EcoR I sites (Figure 2).
Figure 2. The pCR®8/GW/TOPO® Entry vector allows TOPO® TA Cloning® for multiple downstream applications.
|Best: high efficiency, directional TOPO® Cloning Vector Kits||pENTR™/TEV/D-TOPO® Cloning Kit||K253520|
|pENTR™/D-TOPO® Cloning Kit||K240020|
|pENTR™/SD/D-TOPO® Cloning Kit||K242020|
|pENTR™/TEV/D-TOPO® Cloning Kit||K252520|
|Very good: high efficiency TOPO® TA Cloning Vector Kits||pCR®8/GW/TOPO® TA Cloning kit||K252002|
|pCR®8/GW/TOPO® TA cloning kit||K252020|
|pCR®8/GW/TOPO® TA Cloning Kit||K250020|
|Standard restriction cloning Vectors||pENTR™ 1A Dual Selection Vector||A10462|
|pENTR™ 2B Dual Selection Vector||A10463|
|pENTR™ 3C Dual Selection Vector||A10464|
|pENTR™ 4 Dual Selection Vector||A10465|
|pENTR™ 11 Dual Selection Vector||A10467|