Gateway® Recombination Cloning: 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.

Building an Entry clone

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 - pENTR/D-TOPO

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).
pCR8/GW/TOPO
Figure 2. The pCR®8/GW/TOPO® Entry vector allows TOPO® TA Cloning® for multiple downstream applications.

Selection guide for Entry Vectors

RatingKitSKUAdvantages
Best: high efficiency, directional TOPO® Cloning Vector KitspENTR™/TEV/D-TOPO® Cloning KitK253520
  • Directional TOPO® Cloning
  • 5’ TEV sequence for N-terminal tag removal
  • Includes fast-growing competent E. coli that shortens the time for miniprep
 pENTR™/D-TOPO® Cloning KitK240020
  • Directional TOPO® Cloning
 pENTR™/SD/D-TOPO® Cloning KitK242020
  • Directional TOPO® Cloning
  • Vector includes the Shine-Dalgarno sequence for E. coli expression-ready Entry clone
 pENTR™/TEV/D-TOPO® Cloning KitK252520
  • Directional TOPO® Cloning
  • 5’ TEV sequence for N-terminal tag removal
Very good: high efficiency TOPO® TA Cloning Vector KitspCR®8/GW/TOPO® TA Cloning kitK252002
  • Efficient TOPO® TA cloning kitIncludes fast-growing competent E. coli that shortens the time for miniprep
  • Includes plasmid purification components for maximum convenience
 pCR®8/GW/TOPO® TA cloning kitK252020
  • Efficient TOPO® TA cloning with fast-growing competent E. coli that shortens
  • Includes fast-growing competent E. coli that shortens the time for miniprep
 pCR®8/GW/TOPO® TA Cloning KitK250020
  • Efficient TOPO® TA cloning simplifies Entry clone construction
Standard restriction cloning VectorspENTR™ 1A Dual Selection VectorA10462
  • Restriction enzyme cloning vector that produces in-frame (rf = 0), expression ready Entry clones, including both Shine-Dalgarno and Kozak sequences
 pENTR™ 2B Dual Selection VectorA10463
  • Restriction enzyme cloning vector that produce in-frame (rf = +1), expression ready Entry clone
  • Chloramphenical and Kanamycin selection
 pENTR™ 3C Dual Selection VectorA10464
  • Restriction enzyme cloning vector that produce in-frame (rf = +2), expression ready Entry clones
  • Chloramphenical and Kanamycin selection
 pENTR™ 4 Dual Selection VectorA10465
  • Same as pENTR™ 1A Vector except with Nco I instead of Dra I in MCS that produces in-frame (rf = 0), expression-ready Entry clones
  • Chloramphenical and Kanamycin selection
 pENTR™ 11 Dual Selection VectorA10467
  • Same as pENTR™ 1A Vector except with Nsp V instead of Dra I in MCS that produces in-frame (rf = 0), expression-ready Entry clones
  • Chloramphenical and Kanamycin selection