Golden Gate from Part Plasmids — MoClo Assembly Simulator
Digest level-0 part plasmids with a Type IIS enzyme and let the overhangs decide the assembly order.
🔒 Nothing you paste is logged or stored
Paste whole part plasmids — backbone and all — and this runs the reaction the way it happens in the tube. Each part is digested with your Type IIS enzyme (BsaI, BsmBI, BbsI or SapI) and the fragment released is the one left carrying no recognition site, because the site goes out with the donor backbone; the parts are then chained on matching 5′ overhangs rather than on the order you listed them in. The assembly order is therefore an output: you get the excised fragment sizes and overhangs, the junction table, the circular product, and — when the overhangs do not close into a single cycle — the reason why, naming the part and the overhang that matches nothing.
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.
How to use the Golden Gate from Parts tool
- 1Pick the Type IIS enzyme your parts are domesticated for: BsaI, BsmBI, BbsI or SapI.
- 2Paste each part plasmid whole (raw or FASTA), name it, and leave "Circular plasmid" ticked for a level-0 or level-1 part; add up to 24 parts.
- 3Read the excised fragment table — size, left and right overhang for each part.
- 4Read the assembly order the overhangs dictated, its junction table and any warnings, then copy the assembled product or share the link.
Frequently asked questions
How is this different from the Cloning Simulator's Golden Gate mode?
They do the two opposite halves of the method. The Cloning Simulator starts from bare linear fragments and designs the primers that ADD the Type IIS sites and fusion overhangs, assembling in the order you list. This page starts from part plasmids that already carry their sites, cuts them, and works the order out from the overhangs — so the order is an answer rather than an input.
Which fragment does it treat as the part?
The one that comes off with no recognition site left on it. In a correctly domesticated part the two sites face inwards, so the site sits on the piece that is discarded with the backbone — which is also why the assembled product cannot be re-cut. If two different fragments come off site-free, or none does, the tool refuses and says so instead of guessing.
Which enzymes are supported?
BsaI (GGTCTC), BsmBI (CGTCTC), BbsI (GAAGAC) and SapI (GCTCTTC). The first three leave a 4 nt 5′ overhang and SapI a 3 nt one. NEB's variant names such as BsaI-HFv2 and BbsI-HF fold to the parent enzyme; an unrecognised name is rejected with a suggestion rather than quietly substituted, since site, spacer and overhang length are all enzyme-specific.
What happens if my parts do not assemble?
You get the reason, not a blank. Two parts starting or ending with the same overhang means the order is undetermined and both are named; an overhang that matches nothing leaves the chain open and the unplaced parts are listed with every excised fragment size, because a part that came off backbone-sized is the tell that its two sites face outwards; a chain that does not close back to the first part is reported as staying linear.
Does it warn about overhangs that could let a part flip?
Yes, and those warnings fire even on a successful assembly. A self-complementary overhang (AATT, GGCC, ACGT) presents the same sticky end on both strands, so the neighbouring part can ligate either way round; a reverse-complement twin pair such as AATG and CATT makes two junctions indistinguishable. In both cases the product shown is only one of the molecules the reaction gives, and the junction needs redesigning.
My part has an internal BsaI site — will it still work?
No, and that is deliberate. A part with more than one site per strand is refused, because the arc bounded by the extra site is also free of the recognition sequence and would be silently returned as a truncated part. Domesticate the internal site first — the Construct QC Linter's auto-fix removes it with synonymous codon swaps — then re-run.
Does it score how well the overhang set ligates?
No — that is a separate question and a separate page. This tool matches overhangs exactly: two 4-mers either agree or they do not. Whether a set ligates cleanly at scale depends on T4 ligase mismatch data, which the Golden Gate Fidelity tool scores. A set that assembles here can still have a poor predicted fidelity there.
How many parts and how much sequence can I give it?
2 to 24 parts, at most 30,000 bp per part and 200,000 bp in total. The per-part cap is a work limit rather than a biological one: finding the excised fragment costs time proportional to the square of a part's length, and a level-0 or level-1 part plasmid is only a few kb anyway. Assembled products longer than 20,000 bp are truncated in the returned sequence.
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