Restriction Enzyme Cut-Site Finder
Enter a DNA sequence to scan it for recognition sites of common restriction enzymes — EcoRI, BamHI, HindIII, NotI, PstI, SmaI, XhoI, and SalI — showing exactly where each enzyme would cut. Two 3D diagrams compare a blunt cut to a sticky-end (overhang) cut, and charts show how many cut sites each enzyme finds in your sequence.
Reviewed by the ToolNestr Editorial Team — July 2026
| Enzyme | Site | Position | Cut type |
|---|
Blunt cut vs. sticky-end cut
1. Blunt cut (e.g., SmaI)
Both strands cut at the same position — no overhang, a straight break.
2. Sticky-end cut (e.g., EcoRI)
Strands cut at offset positions — a short single-stranded overhang remains.
Cut-site charts
How it works
The core idea in one line: a restriction enzyme is really just a molecular pattern-matcher — wherever its short recognition sequence appears in the DNA, it cuts at that exact, predictable spot, every single time.
Recognition site → search sequence for an exact substring match
e.g., EcoRI recognizes GAATTC anywhere it occurs
Cut position = match start + enzyme-specific offset
each enzyme cuts at a fixed position within its own recognition site
Each restriction enzyme recognizes one specific short DNA sequence, usually 4 to 8 bases long and usually palindromic, and cuts the DNA backbone at a fixed position relative to that sequence whenever it finds a match. Finding all of an enzyme's cut sites in a sequence is as simple as searching for every occurrence of its recognition sequence as an exact substring, then marking the enzyme's known cut offset within each match. Whether the resulting ends are blunt or sticky (with a short single-stranded overhang) depends entirely on whether the enzyme cuts both DNA strands at the same position or at offset positions within the recognition site.
Worked example 1 — a single EcoRI site
Given: Sequence: 5'-AAGAATTCTT-3'. Scanning for EcoRI's recognition site, GAATTC.
EcoRI is one of the most widely used restriction enzymes in molecular cloning, prized for its reliable sticky-end overhangs.
Worked example 2 — two BamHI sites in one sequence
Given: Sequence: 5'-GGATCCAAAGGATCC-3'. Scanning for BamHI's recognition site, GGATCC.
A restriction enzyme cuts at every occurrence of its recognition sequence, not just the first one — this is exactly why longer DNA sequences can be cut into many fragments by a single enzyme.
Common restriction enzymes and their recognition sites
All are palindromic 6-8 base recognition sequences (except SmaI's blunt-cutting 6-base site).
| Enzyme | Recognition site (cut ^) | End type |
|---|---|---|
| EcoRI ★ | G^AATTC | Sticky (5' overhang) |
| BamHI | G^GATCC | Sticky (5' overhang) |
| HindIII | A^AGCTT | Sticky (5' overhang) |
| SmaI | CCC^GGG | Blunt |
| NotI | GC^GGCCGC | Sticky (5' overhang) |
★ Reference row (worked example 1). NotI's 8-base recognition site is much rarer statistically than a 6-base site, making it useful for cutting DNA into very large fragments.
Where restriction enzymes actually matter
🧬 Molecular cloning
Restriction enzymes cut both a gene of interest and a plasmid vector with matching sticky ends, allowing the gene to be inserted precisely into the vector for expression or further study.
🔬 RFLP analysis and DNA fingerprinting
Restriction Fragment Length Polymorphism analysis cuts DNA samples with a chosen enzyme and compares the resulting fragment size patterns, historically used in forensics and paternity testing before modern sequencing became routine.
🧫 Verifying plasmid constructs
After cloning a gene into a plasmid, researchers commonly run a restriction digest ('diagnostic digest') and check the resulting fragment sizes on a gel to confirm the insert is present and correctly oriented.
🦠 Bacterial defense systems
Restriction enzymes exist in bacteria specifically to cut and destroy invading viral (phage) DNA, while the bacterium's own DNA is protected from cutting by a matching methylation system — the original biological purpose these enzymes evolved for.
Common misconceptions
"Every restriction enzyme cuts DNA in exactly the middle of its recognition site."
Cut position varies by enzyme — some cut symmetrically in the middle (like SmaI, giving blunt ends), while many others cut asymmetrically near one end of the site (like EcoRI and BamHI), producing sticky-end overhangs instead.
"A restriction enzyme only cuts a sequence once, no matter how long the DNA is."
An enzyme cuts at every single occurrence of its recognition sequence in the DNA — a longer sequence with multiple matching sites will be cut into that many more fragments.
"Sticky ends from different enzymes can always be joined together."
Sticky ends can only reliably re-anneal with a complementary overhang of the same sequence and length — an EcoRI sticky end generally can't pair directly with a BamHI sticky end, since their overhangs are different sequences.
"Restriction enzymes only exist as laboratory tools, not in nature."
Restriction enzymes are naturally occurring bacterial proteins, evolved specifically as a defense mechanism against bacteriophage (viral) DNA — their use as laboratory tools came later, after scientists discovered and purified them.
Formula sources & further reading
The formulas here are standard, traceable to:
- • OpenStax, Biology 2e — Chapter 17, "Biotechnology and Genomics" (free, peer-reviewed). openstax.org
- • NEB (New England Biolabs) — standard reference for restriction enzyme recognition sites and cut positions.
- • Alberts et al., Molecular Biology of the Cell — Recombinant DNA technology chapter.
Recognition sites and cut positions follow standard published enzyme specifications. Scans the entered strand only, for exact substring matches.
How to use this calculator
Enter a DNA sequence
Type a sequence using only the letters A, T, C, and G.
Review all matches
Every recognition site found across 8 common enzymes is listed with its position.
Note the cut type
Each match shows whether that enzyme leaves sticky or blunt ends.
Related tools
Frequently asked questions
What is a restriction enzyme?
A restriction enzyme (restriction endonuclease) is a protein that recognizes a specific short DNA sequence and cuts the DNA at or near that site — originally a bacterial defense mechanism against viral DNA, now a core tool of molecular biology.
Why are restriction sites usually palindromic?
Most restriction enzyme recognition sequences are palindromic, reading the same on both strands when each is read 5' to 3' (like GAATTC, whose complement read 5' to 3' is also GAATTC) — this lets the enzyme, which typically works as a symmetric dimer, cut both strands using the same recognition mechanism.
What is the difference between sticky ends and blunt ends?
Sticky ends result when an enzyme cuts each strand at a different position within the recognition site, leaving a short single-stranded overhang that can base-pair with a matching sticky end. Blunt ends result when the enzyme cuts both strands at exactly the same position, leaving no overhang.
Why do molecular biologists use restriction enzymes?
They're essential for cutting DNA at precise, predictable locations — used for cloning genes into plasmids, building recombinant DNA constructs, and in diagnostic techniques like RFLP (restriction fragment length polymorphism) analysis.
Can the same restriction enzyme cut a sequence more than once?
Yes — if its recognition sequence appears at multiple locations in the DNA, the enzyme cuts at every occurrence, producing multiple DNA fragments rather than just two.