Golden Gate assembly plan

A Golden Gate reaction drawn as the graph it is: which parts join at which fusion site, in the order the overhangs force.

What it doesCopy Anchor Link

A Type IIS enzyme cuts outside its own recognition site, so it leaves a 4 bp overhang whose sequence is the part's, not the enzyme's. Two parts can be adjacent only if one's right overhang matches the other's left, which means the arrangement is a property of the parts you supplied rather than of the order you listed them in, and the whole reaction can run in one tube in one step.

This is the graph of that arrangement for the construct you have selected. Each level is one assembly step, an edge is a part going into the reaction below it, and the edge is labelled with the fusion site that makes the join. Reading left to right gives the order the fragments end up in; reading down gives the levels of a hierarchical build.

When you'd use thisCopy Anchor Link

• Before ordering parts. A shared or transposed overhang gives a plausible part list and a construct in the wrong order.
• When designing a MoClo-style level. The levels are the shape of the work: which sub-assemblies have to exist before the final reaction can be set up.
• When the reaction produces something unexpected. The graph shows which junction did something you did not intend.
• To check the acceptor joined where you think. A backbone that closed at the wrong end is an arrangement error, and it does not show up in a length.

What this computesCopy Anchor Link

• The assembly graph of the selected construct: its levels, the parts on each, and the edges between them, from the simulated digestion and ligation rather than from the supplied order.
• The fusion site on every edge: the 4 bp overhang that joins those two parts, which is the reason the order is forced.
• The construct's own facts: its length, whether it closed circular, and how many parts it consumed.
• The enzyme in force, because the overhangs, and therefore the whole arrangement, are its.

What it does not doCopy Anchor Link

It draws a simulated reaction, not an experiment. Ligation fidelity between similar overhangs, the efficiency of a given overhang set at a given cycling profile, and the rate of misligation are all real and measured in the literature, and none of them is modelled here: a plan that assembles cleanly on screen can still give colonies with the wrong insert. It also draws the selected construct only.