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. 

Selection guide for Entry vectors

Ordering information

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.
     

Building an Entry clone

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 exicision of insert DNA with flanking EcoR I sites (Figure 3).

 

 

Figure 3. The pCR8/GW/TOPO Entry vector allows TOPO TA Cloning for multiple downstream applications.

Selection guide for Entry vectors

Rating Kit Cat. No. Advantages
Vectors for TOPO cloning
BEST
High efficiency, directional TOPO Cloning Vector Kits
pENTR/TEV/D-TOPO Cloning Kit K253520
  • 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 Kit K240020
  • Directional TOPO Cloning
pENTR/SD/D-TOPO Cloning Kit K242020
  • Directional TOPO Cloning
  • Vector includes the Shine-Dalgarno sequence for E. coli expression-ready Entry clone
pENTR/TEV/D-TOPO Cloning Kit K252520
  • Directional TOPO Cloning
  • 5’ TEV sequence for N-terminal tag removal
VERY GOOD
High efficiency TOPO TA Cloning Vector Kits
pCR8/GW/TOPO TA Cloning kit K252002
  • Efficient TOPO TA cloning kitIncludes fast-growing competent E. coli that shortens the time for miniprep
  • Includes plasmid purification components for maximum convenience
pCR8/GW/TOPO TA cloning kit K252020
  • Efficient TOPO TA cloning with fast-growing competent E. coli that shortens
  • Includes fast-growing competent E. coli that shortens the time for miniprep
pCR8/GW/TOPO TA Cloning Kit K250020
  • Efficient TOPO TA cloning simplifies Entry clone construction
Vectors for PC amplification or restriction enzyme cloning
Standard restriction cloning Vectors pENTR 1A Dual Selection Vector A10462
  • 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 Vector A10463
  • Restriction enzyme cloning vector that produce in-frame (rf = +1), expression ready Entry clone
  • Chloramphenical and Kanamycin selection
pENTR 3C Dual Selection Vector A10464
  • Restriction enzyme cloning vector that produce in-frame (rf = +2), expression ready Entry clones
  • Chloramphenical and Kanamycin selection
pENTR 4 Dual Selection Vector A10465
  • 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 Vector A10467
  • 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

Purchasing a premade clone

You can also utilize Gateway technology with a ready-to-use clone from our extensive clone collection. The 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.

See our selection of Ultimate ORF Clones