Gibson Assembly designer
Maintained by the Talindrew team · Last updated
A Gibson Assembly designer takes the fragments you want to join, works out the homologous overlaps between neighbours, and writes the primers whose 5′ tails carry those overlaps. Talindrew's Gibson wizard does this for multi-fragment assemblies with overlap Tm tuning and automatic primer generation, simulates the assembled product against its cloning engine, and runs a pre-flight design check before you commit. A worked example loads with one click, and multi-step builds continue in the cloning workspace, where a Gibson-family primer designer builds the homology arms from the sequences being joined.
Specifications
| Assembly | Multi-fragment Gibson Assembly with homology overlaps between neighbouring fragments |
|---|---|
| Overlaps | Calculated automatically, with overlap Tm tuning |
| Primers | Generated with 5′ homology tails; annealing Tm and full-oligo Tm reported separately |
| Checks | Pre-flight design check before the design is committed |
| Try first | Worked example loads with one click |
| Multi-step builds | Cloning workspace: Gibson, In-Fusion, In vivo and overlap-extension assemblies with a primer designer |
| Price | Free with a Google sign-in; runs in the browser |
How do I design primers for Gibson Assembly?
Each primer has a 3′ region that anneals to its own template and a 5′ tail that copies the end of the neighbouring fragment, so the PCR products share overlaps that the exonuclease exposes and the fragments anneal in order.
- Add the vector and insert sequences in the order they will be joined.
- Let the wizard calculate the overlaps and tune their Tm; adjust the junctions if a fragment boundary must move.
- Review the generated primers: the annealing Tm drives the PCR, the full-oligo Tm is for reference.
- Check the simulated product map and the pre-flight report, then save the primers or the design to a notebook entry.
Designer or protocol: which page do I need?
This page is the design tool. For the bench side, the amounts, ratios, volumes and incubation times from NEB's Gibson Assembly Master Mix protocol, see the Gibson Assembly guide, which also explains how the exonuclease, polymerase and ligase work together.
What overlap length should a Gibson design use?
Match the overlap to the size of the assembly. NEB's Gibson Assembly Master Mix protocol specifies 15 to 25 bp overlaps between fragments for 2 or 3 part assemblies and 20 to 80 bp for 4 to 6 parts, and an overlap Tm at or above 48 °C keeps annealing from becoming the limiting step. Longer overlaps help large assemblies but make primers longer and more prone to secondary structure.
Frequently asked questions
Does it work for NEBuilder HiFi assemblies too?
Yes. HiFi assembly joins fragments through the same kind of homology overlaps, and Talindrew's 5′ overhang builder makes Gibson and HiFi homology arms on any primer, with the annealing Tm and full-oligo Tm reported separately. Follow the kit's own protocol for amounts and incubation.
Is the Gibson Assembly designer free?
Yes. The Gibson wizard and the rest of the cloning suite are free in the browser with a Google sign-in, with nothing to install.
Does it design the primers with the overlap tails?
Yes. It calculates the homology overlaps between neighbouring fragments, tunes their Tm, and generates primers whose 5′ tails carry the overlaps, reporting the annealing Tm and the full-oligo Tm separately.
Related
Try it on your own sequence.
Free to use. No download required. Works in any modern browser.