Cloning Simulator — Gibson & Golden Gate
Assemble fragments and design junction primers for Gibson, Golden Gate or restriction cloning.
Plan a cloning experiment end to end. Paste your fragments (or a vector and insert), pick an assembly method, Gibson/overlap, Golden Gate with a Type IIS enzyme, or classic restriction–ligation, and the simulator builds the assembled construct and designs the junction primers you need to order. Gibson primers add homology to the neighboring fragment, Golden Gate primers add the recognition site and a 4-bp fusion overhang, and restriction mode digests the vector and insert and reconstitutes the sites. Annealing temperatures come from the same nearest-neighbor engine as the primer tools.
▸Circular product · 20 bp overlap · anneal 60 °C
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Fragments are assembled head-to-tail in the order listed; a circular product wraps the last back to the first. Gibson/overlap primers add homology to the upstream neighbor. Golden Gate primers add the Type IIS site and a 4-bp fusion overhang. Restriction–ligation digests the vector and insert with the chosen enzymes and reconstitutes the sites at both junctions. LIC and SLIC model T4-polymerase chew-back, sequence-defined and time-stopped respectively. In-Fusion/CPEC anneals fragments that already carry ≥15 bp of terminal homology and derives their order from those homologies. Verify junctions and reading frame before ordering.
Got the sequence back? Check it against this recipe
Paste the sequence returned by sequencing. The same method and parts from the recipe above are re-run and compared against it, and the result reports whether they match and, where they do not, which junction differs.
Re-derived from the recipe in the simulator above — Gibson / overlap, 0 fragments, compared as a circular molecule.
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Paste the sequence you got back — it is re-derived from the recipe above and compared.
Parts already cloned as plasmids? Assemble them as they are
MoClo-style part plasmids already carry their Type IIS sites, where the simulator above designs primers to add them. Supply the part plasmids and the enzyme (BsaI, BsmBI, BbsI or SapI); each insert is excised and the parts are chained on their fusion overhangs, returning the assembled construct or the junction that blocks it.
The enzyme your parts are domesticated for. NEB's variant names (BsaI-HFv2, BbsI-HF) fold to the parent enzyme; anything else is refused rather than substituted, because site, spacer and overhang length are all enzyme-specific.
Paste at least 2 whole part plasmids. Each needs one BsaI site per strand, both pointing inwards at the part.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
How to use the Cloning Simulator tool
- 1Choose a method: Gibson/overlap, Golden Gate, or restriction–ligation.
- 2Paste the ordered fragments (or a vector + insert) and set the overlap length or enzyme.
- 3Copy the assembled sequence and the designed primers, or export the primers as CSV.
Frequently asked questions
Which assembly methods are supported?
Three: Gibson/overlap (NEBuilder-style homology), Golden Gate using a Type IIS enzyme (BsaI, BbsI or Esp3I/BsmBI) with 4-bp fusion overhangs, and classic restriction–ligation of a vector and insert cut by two enzymes.
How are the assembly primers designed?
Each fragment's annealing region is grown until its nearest-neighbor Tm (SantaLucia 1998) reaches your target. Gibson primers prepend ~20 bp of homology to the upstream neighbor; Golden Gate primers prepend the Type IIS recognition site plus the fusion overhang. The added tail is highlighted.
Does it check Golden Gate overhangs?
Yes. It flags fusion overhangs that are palindromic or reused across junctions, the two most common causes of failed or scrambled Golden Gate assemblies.