In-Fusion Cloning Calculator
Seamless cloning with 15 bp homology arms — verify each junction and get the recombinant sequence.
Paste at least two fragments, in the order you want them joined.
Learn more
What it does
In-Fusion cloning uses a vaccinia DNA polymerase with 3′→5′ exonuclease activity to expose complementary single-stranded overhangs, which anneal and are repaired inside the cell. Functionally it is seamless like Gibson but needs only about 15 bp of homology, which keeps primer synthesis cheap.
How it works
The tool finds the longest exact suffix/prefix match between consecutive fragments and compares it against your minimum. Because the homology is short, the match has to be exact — a single mismatch inside a 15 bp arm is far more damaging than one inside a 40 bp Gibson arm.
Worked example
A 2.9 kb linearised vector and a 700 bp PCR product sharing 15 bp at each end recombine into a 3.6 kb plasmid. If one arm was ordered with a single wrong base, the junction report shows a homology of 0 and that junction fails.
When to use it
Use it for single-insert cloning where 15 bp primers are cheaper than 30 bp ones, for site-directed insertion without a ligation step, and to confirm multi-fragment In-Fusion designs before ordering oligos.
FAQ
- How is In-Fusion different from Gibson?
- Both are seamless and homology based. In-Fusion needs only ~15 bp and relies on a single enzyme with exonuclease activity; Gibson uses three enzymes and typically 20–40 bp of homology.
- Can I clone a PCR product directly into a vector?
- Yes — that is the main use case. Linearise the vector and add 15 bp extensions to your PCR primers that match the vector ends.
- Does the insert need to be gel purified?
- Usually yes for the vector, to remove uncut template. Crude PCR product often works but raises the background.
- What if a junction shows zero homology?
- The fragments do not share an exact end. Check that you pasted both in 5′→3′ orientation and that the primer extension sequences match the intended partner.