mims-harvard 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, 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
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, BbsIGAAGAC) 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.