Gibson Assembly Calculator
Join DNA fragments by homologous overlap — verify every junction and export the assembled sequence.
Paste at least two fragments, in the order you want them joined.
Learn more
What it does
Gibson Assembly joins overlapping DNA fragments in one isothermal reaction: a 5′ exonuclease chews back each end to expose complementary single strands, a polymerase fills the gaps and a ligase seals the nicks. This calculator does the sequence-level design — it measures the homology at every junction and returns the joined product.
How it works
For each adjacent pair the tool finds the longest exact match between the 3′ end of the upstream fragment and the 5′ end of the downstream fragment. That shared block is the homology arm; it is consumed once, so the product is shorter than the sum of the inputs. A junction passes when the arm reaches your minimum — below about 20 bp the overlap will not reliably anneal at 50 °C.
Worked example
Three fragments of 250 bp, 300 bp and 230 bp with 30 bp homology at both junctions assemble to a 700 bp product — 30 bp is lost at each junction. Shorten one arm to 12 bp and that junction turns red while the other still passes, which is the failure mode you would otherwise only discover on a gel.
When to use it
Use it to verify a Gibson design before ordering primers, to predict the product size for a diagnostic digest, and to check assemblies produced by other tools. It does not model secondary structure, repeats or enzyme kinetics — treat the junction report as a design check, not a yield prediction.
FAQ
- How long should Gibson homology arms be?
- 20–40 bp is the normal range. Below 20 bp annealing becomes unreliable; above 40 bp you pay for extra synthesis without much gain. High-GC junctions benefit from the longer end of the range.
- Why is my product shorter than the sum of my fragments?
- Because each homology arm is shared by two fragments and appears only once in the product. With J junctions you lose the total length of the J arms.
- Can I use Gibson to insert a fragment into a vector?
- Yes. Linearise the vector (by PCR or restriction digest) at the insertion point and treat it as one of the fragments, with homology to both ends of the insert.
- Does fragment order matter?
- Yes. Assembly is directional and depends on each fragment sharing homology with its immediate neighbours, so the order you enter is the order in the product.