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Gateway cloning is a recombination-based method for moving a DNA segment between plasmids using phage lambda att sites and a recombinase enzyme mix instead of restriction enzymes and ligase. A gene is first captured in an Entry Clone (BP reaction) and then transferred into any compatible Destination Vector (LR reaction), keeping its orientation and reading frame.
Short answer
Gateway cloning, described in the primary literature as recombinational cloning, uses site-specific recombination to move a DNA segment between vectors in vitro. Two reactions do the work: BP, which builds an Entry Clone, and LR, which transfers the insert into a Destination Vector to make the Expression Clone. Antibiotic markers and a toxic ccdB gene ensure that mainly the intended product grows.
Definition and scope
The method was described by Hartley, Temple and Brasch in Genome Research (2000), authors who worked at Life Technologies, the company that commercialised it. It builds on the site-specific recombination of the bacteriophage lambda integrase family, which the paper describes as conservative (no net gain or loss of nucleotides) and highly specific. Thermo Fisher Scientific, the current vendor, describes Gateway Technology as a way of cloning without restriction enzymes, ligase or subcloning steps.
It is a cloning and subcloning workflow, not a sequence-editing method. For the wider picture of how a gene gets into a plasmid, including restriction-ligation alternatives, see the CASRAI guide to gene cloning; for reading the vector itself, see the guide to the plasmid map.
How Gateway cloning works in practice
The lambda recombination system
Two reversible reactions of the lambda system are used. The first, attB x attP giving attL + attR, is mediated by integrase (Int) and integration host factor (IHF). The second, attL x attR giving attB + attP, needs Int, IHF and excisionase (Xis). Which direction runs is set by which proteins and sites are supplied. In the commercial format, the BP Clonase mix contains Int and IHF, and the LR Clonase mix adds Xis.
BP reaction: building the Entry Clone
The gene of interest is amplified by PCR with primers carrying 25 bp attB1 (forward) and attB2 (reverse) sites. The attB-flanked product is combined with a donor vector carrying attP sites and BP Clonase, which yields an Entry Clone with the gene between attL1 and attL2. attB1 recombines with attP1 but not attP2, which is what preserves the insert’s orientation. Thermo Fisher also lists TOPO vectors and pre-made ORF collections as routes to an Entry Clone.
LR reaction: moving the gene into a Destination Vector
The Entry Clone is mixed with a Destination Vector (attR1, ccdB, attR2) and LR Clonase. The product is the Expression Clone, with the gene flanked by attB sites. Because one Entry Clone can be sent into many different Destination Vectors in parallel, the same gene can be tested in several expression systems; see the guide to protein expression in E. coli for what usually differs between hosts.
Selection: why mainly the right clone grows
Hartley et al. describe two selection layers. First, the Entry Clone is kanamycin resistant while the Destination Vector is ampicillin resistant, so the markers differ. Second, the Destination Vector carries the F-plasmid ccdB gene, which inhibits E. coli growth, so cells that keep an intact Destination Vector do not form colonies. To propagate ccdB-containing vectors, the authors isolated an E. coli strain, DB3.1, with a gyrA462 mutation that gives resistance to ccdB.
Reaction map: BP versus LR
| Feature | BP reaction | LR reaction |
|---|---|---|
| Substrates | attB-flanked PCR product + attP donor vector | Entry Clone (attL1/attL2) + Destination Vector (attR1/attR2) |
| Enzyme mix | BP Clonase (Int + IHF) | LR Clonase (Int + IHF + Xis) |
| Product | Entry Clone | Expression Clone |
| Antibiotic resistance of the product (Hartley et al.) | Kanamycin (Entry Clone) | Ampicillin (Destination Vector backbone) |
Source: Hartley, Temple and Brasch (2000), Genome Research 10(11):1788-1795.
Where it matters for labs and clinics
Manufacturer claims versus what the primary paper reports
Thermo Fisher states that Gateway reactions reach up to 95% recombination efficiency, compared with about 50% for restriction subcloning, and take roughly 65 minutes against up to 24 hours (excluding miniprep) for conventional subcloning. These are the manufacturer’s own figures. The original paper adds points that a supplier page does not list:
- Insert size. Efficiency of the in vitro reactions decreases as the DNA gets larger, judged by colony counts. A 10 kb PCR product has been cloned with BP (reported as unpublished data in the paper).
- Cryptic attB sites. attB sites are only 25 bp, so a functional site could occur in genomic DNA; the authors speculate that the effective size is in the range of 12-16 bp.
- Host-dependent expression. Expression varied by protein and host system in the paper’s tests (some proteins expressed in insect cells but not E. coli).
- Remaining sites. Recombination sites remain in the final clone; the paper reports that fusions across the attB1 site expressed efficiently.
Pre-run checklist for lab staff
- Record which antibiotic goes with each clone type: kanamycin for Entry Clones, ampicillin for the Destination Vector backbone, as described by Hartley et al.
- Propagate ccdB-containing Destination Vectors only in a ccdB-resistant strain such as DB3.1; a standard strain is inhibited by ccdB.
- Keep the attB1/attP1 and attB2/attP2 pairing in mind when designing primers, since it sets the insert’s orientation.
- Confirm the final construct by digest or sequencing as for any cloning route; see the guides to restriction enzyme digest setup and Sanger sequencing.
Common misunderstandings
- Gateway is scarless. The 25 bp att sites remain in the expression clone, so the junctions are not seamless the way some assembly methods are.
- ccdB is in the Entry Clone. In the source design, ccdB sits in the Destination Vector between attR1 and attR2 and is replaced by the insert during LR.
- BP and LR use different enzymes. Both mixes contain Int and IHF; only the LR mix adds Xis.
- Vendor efficiency figures are universal. The 95% and 65-minute figures are the manufacturer’s claims; the original paper reports efficiency falling as insert size grows.
FAQ
What is Gateway cloning?
A recombination-based cloning system that uses lambda att sites and Clonase enzyme mixes to move DNA between vectors without restriction enzymes or ligase.
What do BP and LR stand for?
They are the names of the two reactions, named for the sites they combine: BP combines attB and attP; LR combines attL and attR.
What does ccdB do in Gateway vectors?
It is a gene whose product inhibits E. coli growth, so it acts as a negative selection marker against Destination Vectors that did not recombine.
Sources
- Hartley JL, Temple GF, Brasch MA. DNA Cloning Using In Vitro Site-Specific Recombination. Genome Research 2000;10(11):1788-1795. doi:10.1101/gr.143000. PubMed Central full text, checked 2026-10-29.
- Thermo Fisher Scientific, Gateway Cloning Technology (manufacturer page), checked 2026-10-29.
