Restriction Enzyme Recognition Sites & Cut Table
A restriction enzyme recognizes a specific short DNA sequence — its recognition site — and cuts the double helix there. This table lists 49 commonly used enzymes with their recognition sequences (5′→3′), the cut position on the top strand (marked ▾), and whether they leave blunt or sticky ends.
| Enzyme | Recognition site (5′→3′) | Length | Ends | Optimal buffer | Temp | Star activity | Methylation blocked |
|---|---|---|---|---|---|---|---|
| AflII | C▾TTAAG | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | — | None |
| AgeI | A▾CCGGT | 6 bp | 5′ overhang (4) | rNEBuffer 1.1 | 37°C | Prone | CpG |
| AluI | AG▾CT | 4 bp | Blunt | rCutSmart | 37°C | — | None |
| ApaI | GGGCC▾C | 6 bp | 3′ overhang (4) | rCutSmart | 37°C | — | Dcm, CpG |
| ApoI | R▾AATTY | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 50°C | Prone | None |
| AscI | GG▾CGCGCC | 8 bp | 5′ overhang (4) | rCutSmart | 37°C | — | CpG |
| AvrII | C▾CTAGG | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | — | None |
| BamHI | G▾GATCC | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | Prone | None |
| BglII | A▾GATCT | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | — | None |
| BspEI | T▾CCGGA | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | — | Dam, CpG |
| BstBI | TT▾CGAA | 6 bp | 5′ overhang (2) | rCutSmart | 65°C | — | CpG |
| BstYI | R▾GATCY | 6 bp | 5′ overhang (4) | rCutSmart | 60°C | — | None |
| ClaI | AT▾CGAT | 6 bp | 5′ overhang (2) | rCutSmart | 37°C | — | Dam, CpG |
| DdeI | C▾TNAG | 5 bp | 5′ overhang (3) | rCutSmart | 37°C | — | None |
| DraI | TTT▾AAA | 6 bp | Blunt | rCutSmart | 37°C | — | None |
| EagI | C▾GGCCG | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | — | CpG |
| EcoRI | G▾AATTC | 6 bp | 5′ overhang (4) | Unique buffer | 37°C | Prone | CpG |
| EcoRV | GAT▾ATC | 6 bp | Blunt | rNEBuffer 3.1 | 37°C | Prone | CpG |
| FseI | GGCCGG▾CC | 8 bp | 3′ overhang (4) | rCutSmart | 37°C | — | Dcm, CpG |
| HaeIII | GG▾CC | 4 bp | Blunt | rCutSmart | 37°C | — | None |
| HindIII | A▾AGCTT | 6 bp | 5′ overhang (4) | rNEBuffer 2.1 | 37°C | Prone | None |
| HinfI | G▾ANTC | 5 bp | 5′ overhang (3) | rCutSmart | 37°C | — | CpG |
| HpaI | GTT▾AAC | 6 bp | Blunt | rCutSmart | 37°C | Prone | CpG |
| KpnI | GGTAC▾C | 6 bp | 3′ overhang (4) | rNEBuffer 1.1 | 37°C | Prone | None |
| MboI | ▾GATC | 4 bp | 5′ overhang (4) | rCutSmart | 37°C | — | Dam |
| MluI | A▾CGCGT | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | — | CpG |
| MspI | C▾CGG | 4 bp | 5′ overhang (2) | rCutSmart | 37°C | — | None |
| NcoI | C▾CATGG | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | — | None |
| NdeI | CA▾TATG | 6 bp | 5′ overhang (2) | rCutSmart | 37°C | — | None |
| NheI | G▾CTAGC | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | Prone | CpG |
| NotI | GC▾GGCCGC | 8 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | — | CpG |
| NsiI | ATGCA▾T | 6 bp | 3′ overhang (4) | rNEBuffer 3.1 | 37°C | — | None |
| PacI | TTAAT▾TAA | 8 bp | 3′ overhang (2) | rCutSmart | 37°C | — | None |
| PmeI | GTTT▾AAAC | 8 bp | Blunt | rCutSmart | 37°C | — | CpG |
| PstI | CTGCA▾G | 6 bp | 3′ overhang (4) | rNEBuffer 3.1 | 37°C | Prone | None |
| PvuII | CAG▾CTG | 6 bp | Blunt | rCutSmart | 37°C | Prone | None |
| SacI | GAGCT▾C | 6 bp | 3′ overhang (4) | rCutSmart | 37°C | — | CpG |
| SalI | G▾TCGAC | 6 bp | 5′ overhang (4) | rNEBuffer 3.1 | 37°C | Prone | CpG |
| SbfI | CCTGCA▾GG | 8 bp | 3′ overhang (4) | rCutSmart | 37°C | Prone | None |
| ScaI | AGT▾ACT | 6 bp | Blunt | rNEBuffer 3.1 | 37°C | Prone | None |
| ScrFI | CC▾NGG | 5 bp | 5′ overhang (1) | rCutSmart | 37°C | Prone | Dcm, CpG |
| SmaI | CCC▾GGG | 6 bp | Blunt | rCutSmart | 37°C | — | CpG |
| SpeI | A▾CTAGT | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | Prone | None |
| SphI | GCATG▾C | 6 bp | 3′ overhang (4) | rCutSmart | 37°C | Prone | None |
| StuI | AGG▾CCT | 6 bp | Blunt | rCutSmart | 37°C | — | Dcm |
| SwaI | ATTT▾AAAT | 8 bp | Blunt | rNEBuffer 3.1 | 25°C | — | None |
| TaqI | T▾CGA | 4 bp | 5′ overhang (2) | rCutSmart | 65°C | — | Dam |
| XbaI | T▾CTAGA | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | Prone | Dam |
| XhoI | C▾TCGAG | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | — | CpG |
▾marks the top-strand cut. Overhang length in parentheses. Optimal buffer, incubation temperature, star-activity propensity and methylation sensitivity are from NEB (rNEBuffer 1.1/2.1/3.1/rCutSmart system). "Methylation blocked" lists which of Dam/Dcm/CpG methylation impair cleavage.
Double-digest buffer finder
To cut with two enzymes in a single tube you need one buffer where both are active. Pick two enzymes and this finder chooses the NEB buffer that maximizes the lowerof the two activities, shows each enzyme's % activity in every buffer, and warns you to run a sequential digest if no single buffer keeps both above ~50% (or if their incubation temperatures differ).
Recommended buffer: rNEBuffer 2.1 — EcoRI 100%, BamHI 100% (min 100%).
| Buffer | EcoRI | BamHI | Min |
|---|---|---|---|
| rNEBuffer 1.1 | 25% | 75% | 25% |
| rNEBuffer 2.1best | 100% | 100% | 100% |
| rNEBuffer 3.1 | 50% | 100% | 50% |
| rCutSmart | 50% | 100% | 50% |
- EcoRI is prone to star activity; avoid long digests / excess units / glycerol >5% in a shared-buffer double digest.
- Check template methylation (EcoRI blocked by CpG); use methylation-free DNA (e.g., dam-/dcm- strain) if needed.
- BamHI is prone to star activity; avoid long digests / excess units / glycerol >5% in a shared-buffer double digest.
- BamHI is not heat-inactivable; use column cleanup between steps if doing a sequential digest.
Blunt vs sticky ends
When an enzyme cuts both strands at the same position it produces blunt ends (e.g. SmaI, EcoRV). When it cuts the two strands at offset positions it leaves short single-stranded overhangs — sticky or cohesive ends. A 5′ overhang (e.g. EcoRI) leaves the single strand on the 5′ side; a 3′ overhang (e.g. PstI, KpnI) on the 3′ side. Sticky ends base-pair with complementary overhangs, which makes directional cloning and ligation more efficient.
4-, 6- and 8-cutters
The length of the recognition site determines how often an enzyme cuts. A 4-cutter (4 bp site, e.g. AluI, HaeIII) cuts on average every ~256 bp; a 6-cutter (e.g. EcoRI, BamHI) about every ~4 kb; an 8-cutter (e.g. NotI, AscI) very rarely, which is useful when you need a single cut in a large construct.
Frequently asked questions
What is a restriction enzyme?
A restriction enzyme (restriction endonuclease) is a protein that recognizes a specific short DNA sequence — its recognition site — and cuts the double helix at or near that site. Bacteria use them as a defense against viral DNA; molecular biologists use them to cut and join DNA fragments.
What is the difference between blunt and sticky ends?
If an enzyme cuts both strands at the same position it leaves blunt ends. If it cuts the two strands at offset positions it leaves short single-stranded overhangs — sticky (cohesive) ends — that base-pair with complementary overhangs, making ligation more efficient.
What is a 6-cutter?
It refers to the length of the recognition site. A 6-cutter recognizes a 6 bp sequence and so cuts on average every ~4,096 bp; a 4-cutter (4 bp site) cuts much more often (~every 256 bp), and an 8-cutter cuts rarely. Longer sites mean fewer, more specific cuts.
What does the ▾ symbol mean in the recognition site?
It marks where the enzyme cuts the top (5′→3′) strand within the recognition sequence. For example EcoRI G▾AATTC cuts between G and A on the top strand, leaving a 4-base 5′ overhang.
Are these the only restriction enzymes?
No — there are thousands. This is a curated set of enzymes commonly used in cloning. For a comprehensive catalog see a supplier database such as REBASE.
Learn more
Sources
- 1REBASE: a database for DNA restriction and modification: enzymes, genes and genomesRoberts RJ, Vincze T, Posfai J, Macelis D · Nucleic Acids Research 51(D1):D629-D630 · 2023The recognition sequence (5'->3') and top-strand cut position for all 49 rows of the main table, and therefore the Length, Ends (blunt / 5' overhang / 3' overhang) and overhang-size columns, which the page computes from the caret position. Also the FAQ answer 'for a comprehensive catalogue see a supplier database such as REBASE'. The repo's generated 566-enzyme table (src/lib/bio/enzymes-rebase.ts) is parsed at build time from REBASE version 608 (gcg, withrefm and bairoch files, 31 Jul 2026) and carries REBASE's supplier codes, isoschizomers and methylase-site notation; the database itself lives at https://rebase.neb.com.
- 2NEBuffer Activity/Performance Chart with Restriction EnzymesNew England BiolabsThe four right-hand columns of the table — Optimal buffer, Temp, Star activity, Methylation blocked — and the footnote naming the rNEBuffer 1.1 / 2.1 / 3.1 / rCutSmart system. Also the per-buffer % activity values and heat-inactivation figures behind the Double-digest buffer finder. Verified row-by-row against the shipped data: EcoRI (supplied NEBuffer EcoRI/SspI, 25/100*/50/50*, 65C HI, 37C), BamHI (r3.1, 75*/100*/100/100*, HI 'No', 37C), HindIII (r2.1, 25/100/50/50, 80C HI), PstI (r3.1), AluI (rCutSmart, 80C HI), SmaI and ApaI both at 37C (not the classic 25C), TaqI-v2 at 65C with HI 'No'. The chart's Dam/Dcm/CpG columns and its colour legend (Not Sensitive / Impaired / Blocked / ...by Overlapping) are the source of the 'Methylation blocked' column. Star-activity flags for 10 of the 49 were taken from the individual NEB product pages' Product Notes, which are reached from this chart.
- 3Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984Cornish-Bowden A (writing for the Nomenclature Committee of the International Union of Biochemistry, NC-IUB) · Nucleic Acids Research 13(9):3021-3030 · 1985The IUPAC/NC-IUB degenerate base symbols that appear inside recognition sites in the table: N in HinfI G.ANTC, DdeI C.TNAG and ScrFI CC.NGG; R and Y in ApoI R.AATTY and BstYI R.GATCY. This is the recommendation the site's IUPAC-aware matching implements. Do NOT cite the 1970 IUPAC-IUB nucleic-acid symbol recommendations (iupac.qmul.ac.uk/misc/naabb.html) for this — that document assigns B, D and S to specific rare nucleosides (5-bromouridine, dihydrouridine, thiouridine), which is a different and conflicting meaning.
Related tools and references
Tools
Find recognition and cut sites for common restriction enzymes.
Pick two restriction enzymes and get the single buffer that runs both, each enzyme's activity in every buffer, and a straight answer on when to digest sequentially instead.
Predict restriction fragments and see the simulated agarose gel with a ladder.