Restriction Enzyme Recognition Sites & Cut Table
A restriction enzyme recognises 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 | — | CpG |
| ApaI | GGGCC▾C | 6 bp | 3′ overhang (4) | rCutSmart | 25°C | — | 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 | — | Dcm, CpG |
| BstBI | TT▾CGAA | 6 bp | 5′ overhang (2) | rCutSmart | 65°C | — | Dam, CpG |
| BstYI | R▾GATCY | 6 bp | 5′ overhang (4) | rCutSmart | 60°C | — | Dam |
| ClaI | AT▾CGAT | 6 bp | 5′ overhang (2) | rCutSmart | 37°C | — | Dam, CpG |
| DdeI | C▾TNAG | 5 bp | 5′ overhang (3) | rNEBuffer 3.1 | 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 | None |
| EcoRV | GAT▾ATC | 6 bp | Blunt | rNEBuffer 3.1 | 37°C | Prone | Dam |
| FseI | GGCCGG▾CC | 8 bp | 3′ overhang (4) | rCutSmart | 37°C | — | CpG |
| HaeIII | GG▾CC | 4 bp | Blunt | rCutSmart | 37°C | — | Dcm, CpG |
| 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 | — | None |
| HpaI | GTT▾AAC | 6 bp | Blunt | rCutSmart | 37°C | Prone | None |
| KpnI | GGTAC▾C | 6 bp | 3′ overhang (4) | rNEBuffer 1.1 | 37°C | Prone | CpG |
| 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 | — | Dcm |
| NdeI | CA▾TATG | 6 bp | 5′ overhang (2) | rCutSmart | 37°C | — | None |
| NheI | G▾CTAGC | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | Prone | Dam |
| 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 | — | None |
| 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 | — | None |
| 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 | — | CpG |
| ScaI | AGT▾ACT | 6 bp | Blunt | rNEBuffer 3.1 | 37°C | Prone | None |
| ScrFI | CC▾NGG | 5 bp | 5′ overhang (1) | rCutSmart | 37°C | — | Dcm, CpG |
| SmaI | CCC▾GGG | 6 bp | Blunt | rCutSmart | 25°C | — | Dcm, CpG |
| SpeI | A▾CTAGT | 6 bp | 5′ overhang (4) | rCutSmart | 37°C | Prone | Dam |
| SphI | GCATG▾C | 6 bp | 3′ overhang (4) | rCutSmart | 37°C | — | 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 | — | CpG |
| 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 maximises 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.
- 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 recognises a specific short DNA sequence — its recognition site — and cuts the double helix at or near that site. Bacteria use them as a defence 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 recognises 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 catalogue see a supplier database such as REBASE.
Learn more
Related tools and references
Use these related pages when this table raises a practical calculation or workflow question.
Tools
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Selection markers, mechanisms and working concentrations for cloning.
PCR primer design rules for length, GC content and Tm.
Reference table of common cloning and protein expression vectors with backbone size, origin of replication, copy number, selection marker, promoter and fusion tags.