Drybench Plasmid.
Golden Gate assembly online (BsaI, BsmBI)
Assemble parts into a destination with one Type IIS enzyme: the scarless product and overhang junctions, with a refusal for any overhang conflict.
The worked example: EGFP part assembled into a destination with BsaI. The enzyme cuts outside its recognition sequence, so each part is released with defined overhangs and the recognition sites leave on the discarded flanks; the parts and the opened backbone join by their overhangs into one scarless circle.
Inputs
- Enzyme: BsaI, a Type IIS enzyme that cuts outside its recognition sequence.
- Destination: destination (demonstration), 59 bp, circular.
- Part: EGFP part, 746 bp, linear. The coding sequence is NCBI U55762.1 679 to 1,398, with Type IIS tails and the chosen overhangs a primer would add.
- Overhangs: AATG, GCTT, distinct and not palindromic.
The released pieces
| Piece | Core (bp) | Left end | Right end |
|---|---|---|---|
| destination (demonstration) fragment 2 (27 bp) | 27 | 5' GCTT | 5' CATT |
| EGFP part fragment 2 (720 bp) | 720 | 5' AATG | 5' AAGC |
The product
A scarless circular product of 755 bp; no recognition site survives in it.
The recognition sites leave on the discarded flanks, so the junctions carry only the designed overhangs.
Junctions
- 5' CATT to 5' AATG, overhang CATT
- 5' AAGC to 5' GCTT, overhang AAGC
Material conserved: 747 core bases and 8 overhang bases, each overhang once, give 755 bp.
Each part is matched in either orientation, since a part and its reverse complement are one molecule: the assembly does not depend on which strand you supplied. A palindromic overhang, a duplicate overhang (including a part that could follow in either orientation), an unresolved internal site or a cut off a linear end is refused, not warned past. No part is domesticated and no coding sequence changed; no fidelity figure is given, and a designed product is not evidence that the reaction occurred.
Computed when this page was built, from data release f1.e1.p1.
How it is computed
- Golden Gate assembly (BsaI, BsmBI). A Type IIS enzyme (BsaI GGTCTC(1/5) or BsmBI CGTCTC(1/5), offsets checked against Biopython Bio.Restriction) cuts outside its recognition sequence, so a part flanked by two inward-pointing sites is released with two defined 5′ overhangs while the recognition sites stay on the discarded flanks; the opened destination backbone and the parts assemble by their exposed overhangs into one scarless circle, decided on the overhangs and polarity alone. Each part is matched in either orientation, since a part and its reverse complement are one double-stranded molecule, so the assembly does not depend on which strand a part was supplied on; the destination backbone is the reference frame. Overhangs must be 5′, non-palindromic and all distinct, and must close into a single cycle; a palindromic overhang, a part that could follow in either orientation, a duplicate or reverse-complement overhang conflict, an unresolved internal site or a cut off a linear end is refused, never a warning to click past. Recognition sites are scanned on physical strand sequences, including the final circular product and every assembled junction; a newly created site is refused. No part is domesticated and no coding sequence changed automatically; no fidelity or ligation-fidelity figure is given. Material is conserved. The product carries a segment map: which input each run of product bases came from and where, a shared overhang belonging to both the inputs that hold it. A designed product is not evidence that the reaction occurred.Checked against 3 assemblies and 4 refusals: a two-part BsaI and a one-part BsmBI assembly whose parts are built around a chosen scarless product, and the same two-part design with one part supplied on the other strand giving the same product, with Bio.Restriction 1.88 cut positions showing the released overhangs, and no recognition site surviving in the product; a palindromic overhang, a duplicate overhang, an unresolved internal site and a Type IIS cut off a linear end each refused (10 October 2026).
- Restriction sites and cut positions. Recognition sequences and cut positions as published facts, typed by hand (pipeline/plasmid-map/enzymes.csv); every row checked against Biopython 1.88 Bio.Restriction, whose data are REBASE release 404 (Roberts RJ et al. REBASE: a database for DNA restriction and modification. Nucleic Acids Res 2023;51:D629-D630).Every row checked against Biopython 1.88 Bio.Restriction (enzyme data release 404) in its own notation, overhang and cut positions: 223 enzymes on pUC19 and pBR322, 446 cases, cut positions exact, with sites across the origin and 6 double digests (2026-10-06).
Limits
- For research and education only, and not medical advice: plasmid maps and sequence assembly designs. A designed product is not evidence that the reaction occurred; nothing here predicts expression or how well a reaction will work.
- Enzyme sites and cut positions are from a hand-curated table of 223 enzymes (version 1, built 2026-10-06); it may differ from a vendor's list, so check NEBcutter before a critical digest. Star activity, Type IIB and nicking enzymes are not modelled.