| 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. |
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
tu run DNA_gibson_design '{"operation":"gibson_design",
"fragments":["ATGGCG...GAGGAC","GAGGAC...GGCAAG","GGGCAAG...ATCCT"],
"overlap_length":20}'
For each fragment it returns left_overlap, , and (the fragment extended with the homology arms you'd add to your PCR primers — hand these to ).
