tooluniverse-molecular-cloning

name	tooluniverse-molecular-cloning
description	Molecular cloning assembly design — Gibson Assembly (overlap design for seamless multi-fragment joining) and Golden Gate Assembly (Type IIS / BsaI / BbsI design with unique 4-bp fusion overhangs). Use when you need to plan how to join DNA fragments into a construct, design assembly overlaps/overhangs, or decide between cloning methods. Covers the domestication (internal-site removal), overhang-uniqueness, and overlap-Tm rules. For PCR primers to generate the fragments, see tooluniverse-primer-design.
disable-model-invocation	true

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.

Step 1 — Gibson Assembly