Drybench Plasmid.
Restriction cloning: ligate an insert into a vector online
Cut a vector and an insert with chosen enzymes and ligate the recombinant circle, with its junctions, every orientation that anneals and any qualification.
The worked example: EGFP insert ligated into pUC19 opened at EcoRI + HindIII. The vector and the insert are each cut with the chosen enzymes, then the backbone and the insert fragment are joined into a recombinant circle. The ends must anneal on their exposed bases and polarity, never the enzyme name.
Walk through a vector, insert and diagnostic digest
Inputs
- Vector: pUC19 (NCBI L09137.2), 2,686 bp, circular.
- Insert: EGFP insert, 738 bp, linear. The coding sequence is NCBI U55762.1 679 to 1,398, with a 9 and a 9 base flank carrying the recognition sites a primer would add.
- Enzymes: EcoRI + HindIII, applied to both the vector and the insert.
Computed on unmethylated (a PCR product or synthetic DNA): set the methylation of the DNA you will cut in the map before you rely on a digest.
The digest
Vector fragments
| Fragment | Core (bp) | Left end | Right end |
|---|---|---|---|
| pUC19 fragment 1 (47 bp) | 47 | 5' AATT | 5' AGCT |
| pUC19 fragment 2 (2631 bp) | 2,631 | 5' AGCT | 5' AATT |
Insert fragments
| Fragment | Core (bp) | Left end | Right end |
|---|---|---|---|
| EGFP insert fragment 1 (4 bp) | 4 | blunt | 5' AATT |
| EGFP insert fragment 2 (722 bp) | 722 | 5' AATT | 5' AGCT |
| EGFP insert fragment 3 (4 bp) | 4 | 5' AGCT | blunt |
The backbone is the pUC19 fragment 2 (2631 bp); the insert fragment is the EGFP insert fragment 2 (722 bp).
The product
A circular recombinant of 3,361 bp. The insert is carried as supplied, on the strand you gave.
Only one orientation anneals: the clone is directional.
Junctions
- 5' AATT to 5' AATT, overhang AATT
- 5' AGCT to 5' AGCT, overhang AGCT
Material conserved: 3,353 core bases and 8 overhang bases, each overhang once, give 3,361 bp.
A designed product is not evidence that the reaction occurred. Nothing here is repaired, filled in, phosphorylated, blunted or mutated, and no ligation efficiency, vector to insert ratio or transformation outcome is given. The products shown are the one-insert circles whose ends anneal: vector self-ligation, circles with more than one insert and concatemers are not enumerated. This example takes the longest enzyme-ended fragment on each side by default.
A single-enzyme clone, where both orientations anneal
The same EGFP insert into pUC19, cut with BamHI alone. Both insert ends expose the same overhang, so the insert anneals either way round and both products are shown rather than one being chosen.
Inputs
- Vector: pUC19 (NCBI L09137.2), 2,686 bp, circular.
- Insert: EGFP insert, 738 bp, linear. The coding sequence is NCBI U55762.1 679 to 1,398, with a 9 and a 9 base flank carrying the recognition sites a primer would add.
- Enzymes: BamHI, applied to both the vector and the insert.
Computed on unmethylated (a PCR product or synthetic DNA): set the methylation of the DNA you will cut in the map before you rely on a digest.
The digest
Vector fragments
| Fragment | Core (bp) | Left end | Right end |
|---|---|---|---|
| pUC19 | 2,682 | 5' GATC | 5' GATC |
Insert fragments
| Fragment | Core (bp) | Left end | Right end |
|---|---|---|---|
| EGFP insert fragment 1 (4 bp) | 4 | blunt | 5' GATC |
| EGFP insert fragment 2 (722 bp) | 722 | 5' GATC | 5' GATC |
| EGFP insert fragment 3 (4 bp) | 4 | 5' GATC | blunt |
The backbone is the pUC19; the insert fragment is the EGFP insert fragment 2 (722 bp).
The product
A circular recombinant of 3,412 bp. The insert is carried as supplied, on the strand you gave.
The ends permit the insert in either orientation, so both products are shown; choose the orientation your design intends.
Junctions
- 5' GATC to 5' GATC, overhang GATC
- 5' GATC to 5' GATC, overhang GATC
Material conserved: 3,404 core bases and 8 overhang bases, each overhang once, give 3,412 bp.
A designed product is not evidence that the reaction occurred. Nothing here is repaired, filled in, phosphorylated, blunted or mutated, and no ligation efficiency, vector to insert ratio or transformation outcome is given. The products shown are the one-insert circles whose ends anneal: vector self-ligation, circles with more than one insert and concatemers are not enumerated. This example takes the longest enzyme-ended fragment on each side by default.
Computed when this page was built, from data release f1.e1.p1.
How it is computed
- Restriction cloning. A vector and an insert are each cut by the chosen enzymes (the restriction and digest methods above, under the host's dam and dcm marks); the chosen backbone and insert fragments are ligated into a circle when their exposed ends anneal, decided on the exposed bases and polarity alone (the double-stranded end method above), never on the enzyme name or a coordinate. The insert is tried both ways and every orientation that anneals is shown, so a symmetric or blunt join is surfaced as ambiguous, not resolved silently. Material is conserved: the product length is the sum of the fragments' top-strand lengths less each shared overhang counted once. The products shown are the one-insert circles whose ends anneal: vector self-ligation, circles carrying more than one insert and concatemers are not enumerated, so the list is not every product a ligation can give. The worked example takes the longest enzyme-ended fragment on each side by default. No end is repaired, filled in, phosphorylated, blunted or mutated; no ligation efficiency, vector:insert ratio or transformation outcome is given; a cut off a linear end refuses. 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 clones and 2 refusals worked by hand: a directional EcoRI/HindIII clone, single-enzyme (BamHI) and blunt (SmaI) clones that permit both insert orientations and so are shown as two products, incompatible ends refused and a Type IIS cut off a linear end refused. Each hand product is round-tripped by Biopython 1.88 Bio.Restriction (it digests back into the insert and backbone cores); material conserved on every product (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).
- Digest fragments. Fragments between consecutive top-strand cuts, across the origin of a circular plasmid; lengths in base pairs on the top strand, as Biopython and NEBcutter report them.Fragments checked against Biopython 1.88 Bio.Restriction on 446 single digests and 6 double digests of pUC19 and pBR322, lengths exact (2026-10-06).
- dam and dcm methylation of sites. McClelland M, Nelson M, Raschke E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 1994;22:3640-3659, Table I.Checked against 19 cases worked by hand from McClelland et al. 1994, Table I (one per outcome, flank and origin overlaps, fully methylated DpnI and host settings, plus hemi-site sensitivity entries for other enzymes; not a hemimethylated-DpnI model), and the cited blocked examples on pUC19 (MboI) and pBR322 (EcoRII), outcomes exact (2026-10-06). Which enzymes need a Table I row (82 whose site can carry a dam or dcm methylated base, 57 of them with a row) checked by a second implementation sharing no code with the engine (2026-10-17); whether Table I lists the other 25 has not been checked.
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.
- Methylation blocking is called only where McClelland et al. (1994) record it; elsewhere a site reads 'may be blocked: sensitivity not recorded', which means unknown, never 'cuts'. 25 of the 82 enzymes whose site can carry a dam or dcm methylated base have no row in this app's copy of Table I, and whether Table I lists them has not been checked.