Paste a DNA sequence to see where over 180 common restriction enzymes cut it. Filter for unique cutters or enzymes that don't cut, view a site map, and tick enzymes to get digest fragment sizes and a virtual gel for linear or circular DNA.
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Topology
Site map
Enzymes
Tick enzymes to digest the sequence below. Positions are the last base before the top-strand cut.
Digest: fragment sizes
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How to use the Restriction Enzyme Finder
Paste your DNA (plain, numbered or FASTA). Choose Linear for PCR products and fragments, Circular for plasmids.
Pick what to show: unique cutters are best for cloning, non-cutters tell you which enzymes leave an insert intact.
Narrow the list by site length, end type or enzyme name.
Tick one or more enzymes in the table or click labels on the map to simulate a digest.
Read the fragment sizes and compare the virtual gel with your real one, then copy or download the list.
How restriction sites and fragments are calculated
Type II restriction enzymes recognize a short DNA sequence, usually 4 to 8 bases, and cut both strands at fixed positions. Most sites are palindromes: the top strand read 5′→3′ is the same as the bottom strand read 5′→3′ (GAATTC for EcoRI). For non-palindromic sites, including Type IIS enzymes such as BsaI that cut outside their site, the tool also searches the reverse complement so that sites on the opposite strand are found.
For a linear molecule of length L with cuts at positions c1 < c2 < … < ck, the fragments are c1, c2 − c1, …, L − ck base pairs: always k + 1 pieces. A circular molecule with k cuts gives k pieces, and the last one wraps from ck back around to c1. In random DNA a specific 4-base site appears about once every 256 bp, a 6-base site once every 4,096 bp and an 8-base site once every 65,536 bp.
Common cloning enzymes
Enzyme
Site (^ = cut)
Ends
Notes
EcoRI
G^AATTC
5′ overhang AATT
Classic MCS enzyme
BamHI
G^GATCC
5′ overhang GATC
Ends compatible with BglII and BclI
HindIII
A^AGCTT
5′ overhang AGCT
Often paired with EcoRI for directional cloning
XhoI
C^TCGAG
5′ overhang TCGA
Ends compatible with SalI
NotI
GC^GGCCGC
5′ overhang GGCC
8-base rare cutter
PstI
CTGCA^G
3′ overhang TGCA
Example of a 3′ overhang
KpnI / Acc65I
GGTAC^C / G^GTACC
3′ / 5′ overhang
Same site, different cut (neoschizomers)
EcoRV
GAT^ATC
Blunt
Blunt-end cloning
SmaI / XmaI
CCC^GGG / C^CCGGG
Blunt / 5′ overhang
Same site, different ends
BsaI
GGTCTC(1/5)
5′ overhang, 4 nt
Type IIS, used in Golden Gate assembly
Tips for planning a digest
Directional cloning: choose two different unique cutters in the vector's cloning site that are also non-cutters in your insert.
Adding sites to primers: check the insert with "Non-cutters" first, then add 4–6 extra bases 5′ of the site so the enzyme can cut near the end of a PCR product.
Diagnostic digests: pick an enzyme combination that gives 2–4 fragments of clearly different sizes between 300 bp and 5 kb, which are easy to resolve on a 1% agarose gel.
Each enzyme's recognition sequence is searched on the top strand and, for non-palindromic sites, also as its reverse complement so sites on the bottom strand are found. Degenerate letters follow IUPAC rules, for example N = any base and R = A or G. The cut position comes from the enzyme's known cleavage point relative to its site.
What do the cut positions mean?
A position is the last base before the cut on the top strand. EcoRI cuts G^AATTC, so a site starting at base 101 is reported at 101. The fragment table uses the same coordinates, starting at 1.
What is a unique cutter and why does it matter?
A unique cutter cuts the sequence exactly once. In a plasmid it opens the circle at a single point, which is what you want for inserting a fragment. Two different unique cutters in a multiple cloning site allow directional cloning.
What are 5′ overhang, 3′ overhang and blunt ends?
When the two strands are cut at different points the enzyme leaves single-stranded sticky ends: a 5′ overhang (EcoRI, BamHI) or a 3′ overhang (PstI, KpnI). If both strands are cut at the same point, as with EcoRV or SmaI, the ends are blunt. The number shows how many bases overhang.
How are circular sequences handled?
In circular mode the search wraps around the end of the sequence, so a site spanning the last and first bases is found. With N cuts a circle gives N fragments; one cut simply linearizes it. Uncut supercoiled plasmids run differently from linear DNA on a real gel.
Does it account for DNA methylation or star activity?
No. The results show every sequence match. In practice Dam or Dcm methylation can block some enzymes (for example XbaI or ClaI sites overlapping GATC), and star activity can create extra cuts under poor buffer conditions, so check the supplier's notes for critical work.
Is my sequence uploaded?
No. The search, map, digest and gel are all computed in your browser; nothing is sent to a server.
Are all the tools on FreeToolCabin tested for quality and accuracy?
Yes! Every tool on our site undergoes automated Q/A testing in real browser environments to ensure high precision, correct output, and zero errors. If you happen to spot a bug or have a suggestion, please contact us at [email protected] and our team will review it right away.