NEBcutter V3.0 is New England Biolabs’ tool for finding the restriction enzyme sites in a DNA sequence. Paste your sequence into the official tool embedded below to map cut sites for Type II and commercially available enzymes, identify enzymes that do not cut, and view fragment sizes — then use the guide underneath to interpret the results.
Open NEBcutter V3.0 in a new tab ↗ (recommended on small screens, or if the tool does not load above).
How to use NEBcutter V3.0
- Enter your sequence. Paste raw DNA or FASTA, or load a GenBank/EMBL accession. Choose linear or circular topology to match your molecule (important for plasmids).
- Run the analysis. NEBcutter digests your sequence in silico against the enzyme set you select (all enzymes, NEB-supplied only, or a custom list).
- Explore the output. View a graphical map of cut sites, a list of enzymes and their positions, enzymes that cut once or not at all, and predicted fragment sizes.
What NEBcutter does
Restriction enzymes recognise specific short sequences and cut the DNA there. Mapping these sites is essential for cloning (choosing where to linearise a vector or excise an insert), diagnostic digests (confirming a construct by its fragment pattern), and RFLP analysis. NEBcutter scans your sequence against a comprehensive enzyme database and reports exactly where each enzyme cuts, so you can pick the right enzyme for the job instead of checking sites by hand.
Reading the results
- Cut-site map. A linear or circular diagram showing each enzyme’s position. Single cutters are highlighted — these are usually what you want for linearising a vector.
- Enzymes that don’t cut. Useful when you need an enzyme guaranteed not to touch your insert.
- Fragment sizes. Predicts the band pattern you should see on a gel after a digest — compare this with your actual gel to confirm a construct.
- ORFs and features. NEBcutter can also display open reading frames to help you avoid cutting within a coding region.
Tips for choosing restriction enzymes
- Prefer single cutters in the vector’s multiple cloning site for directional cloning.
- Check the insert too — the enzyme must not cut within the fragment you want to keep intact.
- Watch for methylation sensitivity (Dam/Dcm); some sites are blocked in DNA from standard E. coli strains.
- Match buffers when planning a double digest so both enzymes are active together.
NEBcutter V3.0 vs V2.0
V3.0 is the current version and replaces the older NEBcutter 2.0 interface (the v2 address now redirects to v3). V3.0 brings an updated enzyme database, a faster interface and improved sequence handling. If you have a bookmark or protocol referencing NEBcutter 2.0, it points to the same analysis run on the modern engine.
NEBcutter as a restriction enzyme & site finder
NEBcutter doubles as a restriction enzyme finder: it lists every enzyme that cuts your sequence and where, so you can pick single cutters or confirm a site is present. To search the other way — from a recognition sequence to the enzymes that read it — use the NEB Enzyme Finder or the REBASE database.
Planning a double digest
For a double digest (cutting with two enzymes at once), first confirm both cut where you want using the map above, then check they share a buffer and temperature with NEB’s Double Digest Finder. If the conditions are incompatible, run a sequential digest (one enzyme, change buffer, then the second). For directional cloning, pick two single cutters that leave incompatible ends.
Frequently asked questions
Is NEBcutter free?
Yes. NEBcutter is provided free by New England Biolabs and runs in your browser; no installation or account is required.
Can I analyse a circular plasmid?
Yes. Select circular topology so cut positions and fragment sizes are calculated correctly across the origin.
Why does an enzyme show no cut sites?
Either the recognition sequence is absent from your DNA, or the site is blocked by Dam/Dcm methylation. NEBcutter flags methylation-sensitive sites.
What is the difference between NEBcutter V3.0 and V2.0?
V3.0 is the current release with an updated database and interface; the old V2.0 URL now redirects to V3.0.
Does the tool work on mobile?
It loads on mobile, but the analysis interface is best used on a larger screen. Use the “open in a new tab” link for more room.
Can I use NEBcutter as a restriction enzyme finder?
Yes — it lists every enzyme that cuts your sequence and where. To find enzymes by recognition site instead, use the NEB Enzyme Finder or REBASE.
How do I plan a double digest?
Confirm both enzymes cut where you want in the map above, then check buffer and temperature compatibility with NEB’s Double Digest Finder; otherwise run a sequential digest.
Related guide: Restriction enzyme cloning: a step-by-step guide →
