Cloning Simulator — Gibson & Golden Gate
Assemble fragments and design junction primers for Gibson, Golden Gate or restriction cloning.
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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.
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Fragments are assembled head-to-tail in order (circular 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 — and In-Fusion/CPEC anneals fragments that already carry ≥15 bp of terminal homology, working the order out for itself. Verify junctions and reading frame before ordering.
Got the sequence back? Check it against this recipe
The simulation above predicts what the reaction should produce. This is the other direction: give it the same method and parts plus the sequence you actually got, and it re-runs the assembly and says whether they match — and if not, which junction is wrong.
Give BARE parts: Gibson homology arms are added by the assembly primers, so the fragments themselves must not already carry them.
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Describes the junction/primer design only. Since the fragments must not carry their arms, it does not change the predicted product length or the verdict.
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Paste the sequence you claim you got, plus the parts the recipe was run with.
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.
More
Guides
Related tools
Predict restriction fragments and see the simulated agarose gel with a ladder.
Find recognition and cut sites for common restriction enzymes.
Pick two restriction enzymes and get the single buffer that runs both, each enzyme's activity in every buffer, and a straight answer on when to digest sequentially instead.