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tooluniverse-molecular-cloning

Molecular cloning, in both directions. DESIGN — Gibson Assembly (overlap design for seamless multi-fragment joining) and Golden Gate Assembly (Type IIS / BsaI / BbsI / Esp3I / BsmBI / SapI design with unique 4-bp fusion overhangs). ANALYSIS — work out what an existing reaction produces: given input plasmid sequences and an enzyme, digest them, join the fragments by their overhangs, and identify features of the product (expressed ORF, gRNA spacer and its target gene). Use when you need to plan how to join DNA fragments into a construct, design assembly overlaps/overhangs, decide between cloning methods, or determine the product of a stated Gibson/Golden Gate reaction. Covers the domestication (internal-site removal), overhang-uniqueness, and overlap-Tm rules. For PCR primers to generate the fragments, see tooluniverse-primer-design.

skills/tooluniverse-molecular-cloning/SKILL.md

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# Molecular Cloning Assembly Design (Gibson & Golden Gate)

Plan how to join DNA fragments into a construct: design the **overlaps** (Gibson) or **Type IIS overhangs** (Golden Gate) and avoid the failures that come from internal sites and non-unique junctions.

## Step 0 — Pick the method

| Use **Gibson Assembly** when | Use **Golden Gate** when |
|---|---|
| A few fragments, **scarless/seamless** junctions anywhere you choose | Many parts, **standardized reusable** parts (MoClo/modular), one-pot |
| You can add ~20–40 bp homology by PCR | You can remove internal BsaI/BbsI sites (domestication) |
| One-off constructs | Combinatorial libraries / repeated assemblies |

Both are sequence-independent (no scar at the junction for Gibson; a 4-bp fusion scar for Golden Gate). For 2–4 unique fragments, Gibson is usually simplest; for libraries or a parts toolkit, Golden Gate.