Type IIS Restriction Enzymes
Type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence. They are useful for many applications, including Golden Gate Assembly. NEB currently offers over 50 Type IIS restriction enzymes. This table allows you to sort our enzymes by feature for easy comparison.
We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.
| Enzyme | Heat Inact. | NEBuffer | Rxn Temp. | Activity at 37°C | Storage Temp. | Recognition Sequence | Recognition Sequence Length | Over- hang Length | Isoschizomers from NEB | Methylation Sensitivity** | Enz. Sub- type | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AcuI | Y | rCutSmart | 37°C | -20°C | CTGAAG(16/14) | 6 | 2 | IIC | ||||
| AlwI | N | rCutSmart | 37°C | -20°C | GGATC(4/5) | 5 | 1 | dam | ||||
| BaeI |  |
Y | rCutSmart | 25°C | 100% |
-20°C | (10/15)ACNNNNGTAYC(12/7) | 7 | 5 & 5 | IIC | ||
| BbsI * | Y | NEBuffer r2.1 | 37°C | -80°C | GAAGAC(2/6) | 6 | 4 | IIT | ||||
| BbsI-HF * | Y | rCutSmart | 37°C | -20°C | GAAGAC(2/6) | 6 | 4 | IIT | ||||
| BbvI |  |
Y | rCutSmart | 37°C | -20°C | GCAGC(8/12) | 5 | 4 | ||||
| BccI | Y | rCutSmart | 37°C | -20°C | CCATC(4/5) | 5 | 1 | |||||
| BceAI | Y | NEBuffer r3.1 | 37°C | -20°C | ACGGC(12/14) | 5 | 2 | CpG | ||||
| BcgI | |
Y | NEBuffer r3.1 | 37°C | -20°C | (10/12)CGANNNNNNTGC(12/10) | 6 | 2 & 2 | dam; CpG | IIC | ||
| BciVI | Y | rCutSmart | 37°C | -20°C | GTATCC(6/5) | 6 | 1 | |||||
| BcoDI | N | rCutSmart | 37°C | -20°C | GTCTC(1/5) | 5 | 4 | BsmAI | CpG | IIT | ||
| BfuAI | |
Y | NEBuffer r3.1 | 50°C | 50% | -20°C | ACCTGC(4/8) | 6 | 4 | BspMI | CpG | |
| BmrI | Y | NEBuffer r2.1 | 37°C | -20°C | ACTGGG(5/4) | 6 | 1 | |||||
| BpmI | |
Y | NEBuffer r3.1 | 37°C | -20°C | CTGGAG(16/14) | 6 | 2 | IIC | |||
| BpuEI | Y | rCutSmart | 37°C | -20°C | CTTGAG(16/14) | 6 | 2 | IIC | ||||
| BsaI-HF®v2 * | Y | rCutSmart | 37°C | -20°C | GGTCTC(1/5) | 6 | 4 | dcm; CpG | IIT | |||
| BsaXI | N | rCutSmart | 37°C | -20°C | (9/12)ACNNNNNCTCC(10/7) | 6 | 3 & 3 | IIC | ||||
| BseRI | Y | rCutSmart | 37°C | -20°C | GAGGAG(10/8) | 6 | 2 | IIC | ||||
| BsgI | |
Y | rCutSmart | 37°C | -20°C | GTGCAG(16/14) | 6 | 2 | IIC | |||
| BsmAI | N | rCutSmart | 55°C | 50% | -20°C | GTCTC(1/5) | 5 | 4 | BcoDI | CpG | ||
| BsmBI-v2 * | Y | NEBuffer r3.1 | 55°C | 10% | -20°C | CGTCTC(1/5) | 6 | 4 | Esp3I | CpG | IIT | |
| BsmFI | Y | rCutSmart | 65°C | 100% | -20°C | GGGAC(10/14) | 5 | 4 | CpG; dcm | IIC | ||
| BsmI | Y | rCutSmart | 65°C | 20% | -20°C | GAATGC(1/-1) | 6 | 2 | IIT | |||
| BspCNI | Y | rCutSmart | 37°C | -20°C | CTCAG(9/7) | 5 | 2 | IIC | ||||
| BspMI | |
Y | NEBuffer r3.1 | 37°C | -20°C | ACCTGC(4/8) | 6 | 4 | BfuAI | |||
| BspQI * | Y | NEBuffer r3.1 | 50°C | 50% | -20°C | GCTCTTC(1/4) | 7 | 3 | SapI | IIT | ||
| BsrDI | Y | NEBuffer r2.1 |
65°C | 50% | -20°C | GCAATG(2/0) | 6 | 2 | IIT | |||
| BsrI | Y | NEBuffer r3.1 | 65°C | 10% | -20°C | ACTGG(1/-1) | 5 | 2 | IIT | |||
| BtgZI * | Y | rCutSmart | 60°C | 50% | -20°C | GCGATG(10/14) | 6 | 4 | CpG | IIC | ||
| BtsCI | Y | rCutSmart | 50°C | 25% | -20°C | GGATG(2/0) | 5 | 2 | ||||
| BtsI-v2 | Y | rCutSmart | 37°C | -20°C | GCAGTG(2/0) | 6 | 2 | IIT | ||||
| BtsIMutI | Y | rCutSmart | 55°C | 50% | -20°C | CAGTG(2/0) | 5 | 2 | IIT | |||
| CspCI | |
Y | rCutSmart | 37°C | -20°C | (11/13)CAANNNNNGTGG(12/10) | 7 | 2 & 2 | IIC | |||
| EarI | Y | rCutSmart | 37°C | -20°C | CTCTTC(1/4) | 6 | 3 | CpG | IIT | |||
| EciI | Y | rCutSmart | 37°C | -20°C | GGCGGA(11/9) | 6 | 2 | CpG | IIC | |||
| Esp3I * | Y | rCutSmart | 37°C | -20°C | CGTCTC(1/5) | 6 | 4 | BsmBI-v2 | CpG | IIT | ||
| FauI | Y | rCutSmart | 55°C | 50% | -20°C | CCCGC(4/6) | 5 | 2 | CpG | |||
| FokI | |
Y | rCutSmart | 37°C | -20°C | GGATG(9/13) | 5 | 4 | dcm; CpG | |||
| HgaI | Y | NEBuffer r1.1 | 37°C | -20°C | GACGC(5/10) | 5 | 5 | CpG | ||||
| HphI | Y | rCutSmart | 37°C | -20°C | GGTGA(8/7) | 5 | 1 | dam; dcm | ||||
| HpyAV | Y | rCutSmart | 37°C | -20°C | CCTTC(6/5) | 5 | 1 | CpG | ||||
| MboII | |
Y | rCutSmart | 37°C | -20°C | GAAGA(8/7) | 5 | 1 | dam | |||
| MlyI | Y | rCutSmart | 37°C | -20°C | GAGTC(5/5) | 5 | 0 | |||||
| MmeI | |
Y | rCutSmart | 37°C | -20°C | TCCRAC(20/18) | 6 | 2 | CpG | IIC | ||
| MnlI | Y | rCutSmart | 37°C | -20°C | CCTC(7/6) | 4 | 1 | |||||
| NmeAIII | |
Y | rCutSmart | 37°C | -20°C | GCCGAG(21/19) | 6 | 2 | IIC | |||
| PaqCI * |
|
Y | rCutSmart | 37°C | -20°C | CACCTGC(4/8) | 7 | 4 | CpG | |||
| PleI | |
Y | rCutSmart | 37°C | -20°C | GAGTC(4/5) | 5 | 1 | CpG | |||
| SapI * | Y | rCutSmart | 37°C | -20°C | GCTCTTC(1/4) | 7 | 3 | BspQI | IIT | |||
| SfaNI | Y | NEBuffer r3.1 | 37°C | -20°C | GCATC(5/9) | 5 | 4 | CpG |
* Cited for use in Golden Gate Assembly according to current literature
** Methylation sensitivity applies to the recognition motif only
Through suppression experiments and published reports, NEB has identified that these enzymes require more than one recognition site on the substrate to cleave optimally. For more information, visit Restriction Enzyme Cleavage: ‘single-site’ enzymes and ‘multi-site’ enzymes.
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