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

bash
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, right_overlap, and with_overlaps (the fragment extended with the homology arms you'd add to your PCR primers — hand these to tooluniverse-primer-design).

Gibson design rules

• Overlap length 15–40 bp (20–25 typical); longer for GC-poor junctions.
• Overlap Tm ≈ 48–65 °C and balanced between junctions.
• Fragment order matters — list fragments in assembly order; the last fragment's 3′ overlaps the first only if you're making a circle (vector).
• Avoid repeats/secondary structure at the junctions (hairpins, direct repeats) → misassembly.
• Unique junctions — if two junctions share homology, fragments can swap; redesign so each overlap is unique.

Step 2 — Golden Gate Assembly

bash
tu run DNA_golden_gate_design '{"operation":"golden_gate_design",
  "parts":["ATGGCG...AAGAAC","CTGAGC...CTGATC","GAGGAG...GTGGTG"],
  "enzyme":"BsaI"}'

Returns parts_with_overhangs: each part's unique 4-bp left_overhang/right_overhang and the full_sequence flanked by the Type IIS recognition sites (e.g. BsaI GGTCTC(N1) … cutting outside its site to leave the 4-bp fusion overhang).

Golden Gate design rules

• Domestication is mandatory. The chosen enzyme's site (BsaI GGTCTC, BbsI GAAGAC) must NOT occur inside any part, or it will be cut internally. Remove internal sites by silent mutation before assembly — check every part.
• Overhangs must be unique and non-palindromic. Each 4-bp fusion site must differ from the others and not equal its own reverse complement, or junctions misligate. The tool assigns unique non-palindromic overhangs; keep them.
• Avoid high-GC or all-AT overhangs; published high-fidelity overhang sets (e.g. Potapov 2018) ligate most cleanly.
• Order is encoded by the overhangs, not by listing order — the 4-bp junctions define assembly.

Step 3 — QC before ordering

scripts/cloning_qc.py screens parts for the problems above: internal BsaI/BbsI sites (Golden Gate), overhang uniqueness/palindromes, and Gibson overlap GC/length — and flags PASS/WARN.

Step 4 — Gotchas (state these)

• Internal Type IIS sites (Golden Gate) — the #1 failure; domesticate every part.
• Non-unique Gibson overlaps or shared homology → fragment swapping / misassembly.
• Repeats and strong secondary structure at junctions reduce efficiency in both methods.
• Overlap Tm imbalance (Gibson) → some junctions form, others don't.
• Generating the fragments still needs primers with the overlaps/overhangs appended — design and QC those in tooluniverse-primer-design (and BLAST for specificity).

Honest limitations

• These tools design the assembly junctions; they do not simulate the full ligation/exonuclease reaction or guarantee efficiency — validate by sequencing the assembled construct.
• No vector-backbone or ORF-frame checking — confirm reading frame and backbone compatibility yourself.

Related skills

• tooluniverse-primer-design — design the PCR primers (with homology arms / Type IIS tails) to make the fragments.
• tooluniverse-sequence-analysis — handle the input sequences.