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 we are in the process of switching all reaction buffers to be BSA-free. Beginning April 2021, NEB will be switching our current BSA-containing reaction buffers (NEBuffer™ 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We anticipate that this switch may take as long as 6 months to complete. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Find more details at www.neb.com/BSA-free.
During this transition period, you may receive product with BSA or rAlbumin-containing buffers. NEB has rigorously tested both and has not seen any difference in enzyme performance when using either buffer. Either buffer can be used with your enzyme. All website content will be switched in April to reflect the changes, although you may not receive the new buffer with your product immediately.
| 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 | 20% | -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 | 20% | -20°C | CGTCTC(1/5) | 6 | 4 | Esp3I | CpG | IIT | |
| BsmFI | Y | rCutSmart | 65°C | 50% | -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 | 25°C | 75% | -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 | 10% | -20°C | GCTCTTC(1/4) | 7 | 3 | SapI | IIT | ||
| BsrDI | Y | NEBuffer r2.1 |
65°C | 30% | -20°C | GCAATG(2/0) | 6 | 2 | IIT | |||
| BsrI | Y | NEBuffer r3.1 | 65°C | 20% | -20°C | ACTGG(1/-1) | 5 | 2 | IIT | |||
| BtgZI * | Y | rCutSmart | 60°C | 75% | -20°C | GCGATG(10/14) | 6 | 4 | CpG | IIC | ||
| BtsCI | Y | rCutSmart | 50°C | 50% | -20°C | GGATG(2/0) | 5 | 2 | ||||
| BtsI-v2 | Y | rCutSmart | 55°C | 75% | -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 | 20% | -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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