Golden Gate Type IIS Overhang and Assembly Inspector
Inspect the existing BsaI assembly example with an EGFP-derived part and a small synthetic destination. The sequence engine models cleavage outside recognition sites and the resulting overhang joins. Its output is a designed assembly under explicit sequence rules, not evidence of ligation fidelity or experimental clone identity.
The existing worked example uses EGFP coding bases 679–1398 from GenBank U55762.1, synthetic BsaI flanks and a demonstration destination. AATG and GCTT are the declared overhangs. The destination is illustrative and is not described as a functional expression vector.
Trace the sequence from recognition to ligation
A Golden Gate design has at least three distinct sequence features: the recognition motif, the cut positions and the overhang presented for joining. These may be close together, but they are not interchangeable. Follow the synthetic flanks through the digest to see which bases are removed and which enter the final product. This makes an unexpected scar or missing base easier to diagnose.
The reference’s destination is intentionally small so the logic of the junctions is visible. It does not claim replication, selection or expression function. The EGFP-derived part carries a cited sequence interval, while the added flanks are original demonstration design. Keeping those sources separate prevents an engineered demonstration from being mistaken for an unchanged database plasmid.
Review uniqueness and biological context
A set of ends may allow an intended circular product, alternative orientations or competing joins depending on its design. Sequence compatibility alone does not quantify the frequency of each product in a reaction. Review overhang uniqueness and retained recognition sites, and keep any ambiguity reported by the engine rather than selecting a preferred output without explanation.
An internal Type IIS site is a sequence-level constraint. Removing it may be straightforward in a permissive linker, more consequential in a coding region and unacceptable in an essential regulatory element. Preserve the reasoning for every domestication change. Experimental assembly and subsequent sequence confirmation remain necessary to establish that the intended construct was obtained, even when the in-silico design is internally consistent.
Equations and variables
Designed assembly length accounting
Lproduct = Σ Lretained pieces − Σ Lshared junction overlap
- Lproduct
- Length of the designed product sequence (bp)
- Lretained pieces
- Sequence retained after the modelled cuts (bp)
- Lshared junction overlap
- Bases represented by both compatible joining ends (bp)
Overhang compatibility
endA = reverse-complement(endB), under the stated end convention
- endA, endB
- Exposed strand sequences after Type IIS cleavage
- end convention
- Polarity and strand orientation used by the engine
Scope and limitations
- Internal recognition sites can fragment a part or destination. Detecting such a site does not automatically provide a biologically acceptable domestication edit.
- Compatible ends establish a possible join under the sequence model, not measured ligation efficiency or mismatch discrimination.
- The example uses BsaI. BsmBI is a supported selectable enzyme, but changing the enzyme requires compatible recognition-site design rather than relabelling unchanged flanks.
Sources and provenance
- GenBank pEGFP-N1 sequence U55762.1Existing source notice: NCBI public domain
Only the documented EGFP coding interval is used with original demonstration flanks.
- Engler et al.: a one-pot Type IIS assembly methodCC BY; article reference
- Plasmid assembly methodsExisting project method and fixture notices
Technical questions
Why can the recognition sites disappear from the product?
Type IIS enzymes cut outside their recognition sequence. A design can place the recognition sites on discarded flanks while retaining the desired joined parts. Whether that happens depends on orientation and cut offsets, so inspect the modelled pieces instead of assuming every Type IIS design is scarless.
Will the tool automatically domesticate an internal site?
No automatic biological repair is implied. A sequence edit that removes a recognition motif can change coding, regulatory or structural information. Choose and justify an edit in its actual context, then repeat the sequence checks and retain the original and revised designs.
Can I substitute BsmBI for BsaI without changing the parts?
Not as a simple label change. Their recognition sequences differ, and the parts must be designed for the selected enzyme. Use the relevant enzyme definition and inspect cleavage orientation, retained sequence and overhangs for the actual input.
Connected workflows
- Virtual restriction digest and fragment sizes
Inspect recognition and cleavage positions in a simpler digest.
- Vector and insert molar-ratio calculation
Compare overlap-based assembly and molecule-amount planning.
- Six-frame translation and ORF inspection
Check the coding consequences of a proposed sequence edit.