Cloning & Assembly Simulator — Gibson, Golden Gate & Restriction
Assemble fragments and design junction primers for Gibson, Golden Gate or restriction cloning.
🔒 Local processing — pasted sequences are not uploaded
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 neighbouring 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-neighbour engine as the primer tools.
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Provide at least two fragments.
Fragments are assembled head-to-tail in order (circular wraps the last back to the first). Gibson/overlap primers add homology to the upstream neighbour; 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. Verify junctions and reading frame before ordering.
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-neighbour Tm (SantaLucia 1998) reaches your target. Gibson primers prepend ~20 bp of homology to the upstream neighbour; 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.