Plasmid MapHow a restriction digest works
A restriction enzyme cuts DNA at a short recognition site. EcoRI, for example, cuts GAATTC between the G and the first A on each strand. Cutting a circular plasmid at n sites gives n fragments, because the ends of the circle join up; cutting a linear molecule at n sites gives n + 1. Each fragment's size is the distance from one cut to the next.
Paste a sequence, choose circular or linear, and enter up to six enzymes. The calculator finds every exact site on both strands, including sites that span the origin of a plasmid, and lists each fragment with its position and the enzymes at its ends.
Worked example: pUC19 with ScaI and PvuII
pUC19 is a 2,686 bp circular cloning vector. ScaI cuts it once, at 1,192, and PvuII cuts it twice, at 55 and 377. The three cuts give three fragments: 1,549 bp, 815 bp and 322 bp, which separate cleanly on a 1% agarose gel. Both enzymes leave blunt ends.
Digest the same sequence as linear DNA and the fragment that crossed the origin becomes two pieces, 1,495 and 54 bp, so the result is four fragments.
Reading the virtual gel
DNA fragments move through an agarose gel at a rate that falls with the logarithm of their size, so the schematic places bands on a log scale from 100 bp to 10 kb. Two fragments of similar size run almost together: EcoRI and BsaI cut pUC19 into 1,364 and 1,322 bp, which a standard 1% gel shows as one thick band or a close doublet. Fragments of exactly the same size always share one band, marked ×2.
Real gels also depend on the agarose percentage, voltage, buffer and loading. Very small fragments are faint or run off the gel. An uncut plasmid runs as supercoiled, nicked and other forms rather than at its linear size, so the gel leaves it out.
Single, double and multiple digests
A double digest cuts with two enzymes at once. Both must work in the same buffer and at the same temperature, so check the supplier's double-digest guidance before you combine them. Methylation matters too: the Dam and Dcm methylation of ordinary E. coli strains blocks some enzymes, and CpG methylation of DNA from mammalian cells blocks others, so a site in the sequence may not cut.
The calculator assumes a complete digest. A partial digest leaves some sites uncut and adds larger bands that span them.
Ends for cloning
Each enzyme's entry shows its end: blunt, or a 5′ or 3′ overhang. EcoRI leaves a 4-base 5′ overhang (AATT) and KpnI a 4-base 3′ overhang. Two fragments ligate efficiently when their overhangs pair; blunt ends join any blunt end. To choose DNA amounts for the ligation, use the ligation calculator. To see an annotated map with every single-cutting enzyme, open the sequence in the Plasmid Map workspace.
Sources: recognition sites and cut positions are REBASE data as distributed with Biopython 1.85 (Bio.Restriction), limited to enzymes listed as supplied by New England Biolabs; fragment sizes match Biopython's site search on pUC19 and random test sequences. pUC19 is NCBI record M77789.2. Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE: a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Research 51, D629–D630 (2023).