Type IIS Restriction Enzymes
| 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 | CutSmart | 37°C | -20°C | CTGAAG(16/14) | 6 | 2 | SAM required | IIC | |||
| AlwI | N | CutSmart | 37°C | -20°C | GGATC(4/5) | 5 | 1 | dam | ||||
| BaeI | Y | CutSmart | 25°C | 20% | -20°C | (10/15)ACNNNNGTAYC(12/7) | 7 | 5 & 5 | SAM required | IIC | ||
| BbsI * | Y | NEBuffer 2.1 | 37°C | -80°C | GAAGAC(2/6) | 6 | 4 | IIT | ||||
| BbsI-HF * | Y | CutSmart | 37°C | -20°C | GAAGAC(2/6) | 6 | 4 | IIT | ||||
| BbvI |  |
Y | CutSmart | 37°C | -20°C | GCAGC(8/12) | 5 | 4 | ||||
| BccI | Y | CutSmart | 37°C | -20°C | CCATC(4/5) | 5 | 1 | |||||
| BceAI | Y | NEBuffer 3.1 | 37°C | -20°C | ACGGC(12/14) | 5 | 2 | CpG | ||||
| BcgI | |
Y | NEBuffer 3.1 | 37°C | -20°C | (10/12)CGANNNNNNTGC(12/10) | 6 | 2 & 2 | dam; CpG; SAM required | IIC | ||
| BciVI | Y | CutSmart | 37°C | -20°C | GTATCC(6/5) | 6 | 1 | |||||
| BcoDI | N | CutSmart | 37°C | -20°C | GTCTC(1/5) | 5 | 4 | BsmAI | CpG | IIT | ||
| BfuAI | |
Y | NEBuffer 3.1 | 50°C | 50% | -20°C | ACCTGC(4/8) | 6 | 4 | BspMI | CpG | |
| BmrI | Y | NEBuffer 2.1 | 37°C | -20°C | ACTGGG(5/4) | 6 | 1 | |||||
| BpmI | |
Y | NEBuffer 3.1 | 37°C | -20°C | CTGGAG(16/14) | 6 | 2 | IIC | |||
| BpuEI | Y | CutSmart | 37°C | -20°C | CTTGAG(16/14) | 6 | 2 | SAM required | IIC | |||
| BsaI * | Y | CutSmart | 37°C | -20°C | GGTCTC(1/5) | 6 | 4 | dcm; CpG | IIT | |||
| BsaI-HF® * | Y | CutSmart | 37°C | -20°C | GGTCTC(1/5) | 6 | 4 | dcm; CpG | IIT | |||
| BsaI-HF®v2 * | Y | CutSmart | 37°C | -20°C | GGTCTC(1/5) | 6 | 4 | dcm; CpG | IIT | |||
| BsaXI | N | CutSmart | 37°C | -20°C | (9/12)ACNNNNNCTCC(10/7) | 6 | 3 & 3 | IIC | ||||
| BseRI | Y | CutSmart | 37°C | -20°C | GAGGAG(10/8) | 6 | 2 | IIC | ||||
| BsgI |  |
Y | CutSmart | 37°C | -20°C | GTGCAG(16/14) | 6 | 2 | SAM required | IIC | ||
| BsmAI | N | CutSmart | 55°C | 50% | -20°C | GTCTC(1/5) | 5 | 4 | BcoDI | CpG | ||
| BsmBI * | Y | NEBuffer 3.1 | 55°C | 20% | -20°C | CGTCTC(1/5) | 6 | 4 | Esp3I | CpG | IIT | |
| BsmFI | Y | CutSmart | 65°C | 50% | -20°C | GGGAC(10/14) | 5 | 4 | CpG; dcm | IIC | ||
| BsmI | Y | CutSmart | 65°C | 20% | -20°C | GAATGC(1/-1) | 6 | 2 | IIT | |||
| BspCNI | Y | CutSmart | 25°C | 75% | -20°C | CTCAG(9/7) | 5 | 2 | SAM required | IIC | ||
| BspMI | |
Y | NEBuffer 3.1 | 37°C | -20°C | ACCTGC(4/8) | 6 | 4 | BfuAI | |||
| BspQI * | Y | NEBuffer 3.1 | 50°C | 10% | -20°C | GCTCTTC(1/4) | 7 | 3 | SapI | IIT | ||
| BsrDI | Y | CutSmart | 65°C | 30% | -20°C | GCAATG(2/0) | 6 | 2 | IIT | |||
| BsrI | Y | NEBuffer 3.1 | 65°C | 20% | -20°C | ACTGG(1/-1) | 5 | 2 | IIT | |||
| BtgZI * | Y | CutSmart | 60°C | 75% | -20°C | GCGATG(10/14) | 6 | 4 | CpG | IIC | ||
| BtsCI | Y | CutSmart | 50°C | 50% | -20°C | GGATG(2/0) | 5 | 2 | ||||
| BtsI | Y | CutSmart | 55°C | 75% | -20°C | GCAGTG(2/0) | 6 | 2 | IIT | |||
| BtsIMutI | Y | CutSmart | 55°C | 50% | -20°C | CAGTG(2/0) | 5 | 2 | IIT | |||
| CspCI | |
Y | CutSmart | 37°C | -20°C | (11/13)CAANNNNNGTGG(12/10) | 7 | 2 & 2 | SAM required | IIC | ||
| EarI | Y | CutSmart | 37°C | -20°C | CTCTTC(1/4) | 6 | 3 | CpG | IIT | |||
| EciI | Y | CutSmart | 37°C | -20°C | GGCGGA(11/9) | 6 | 2 | CpG | IIC | |||
| Esp3I * | Y | CutSmart | 37°C | -20°C | CGTCTC(1/5) | 6 | 4 | BsmBI | CpG | IIT | ||
| FauI | Y | CutSmart | 55°C | 20% | -20°C | CCCGC(4/6) | 5 | 2 | CpG | |||
| FokI | |
Y | CutSmart | 37°C | -20°C | GGATG(9/13) | 5 | 4 | dcm; CpG | |||
| HgaI | Y | NEBuffer 1.1 | 37°C | -20°C | GACGC(5/10) | 5 | 5 | CpG | ||||
| HphI | Y | CutSmart | 37°C | -20°C | GGTGA(8/7) | 5 | 1 | dam; dcm | ||||
| HpyAV | Y | CutSmart | 37°C | -20°C | CCTTC(6/5) | 5 | 1 | CpG | ||||
| MboII | |
Y | CutSmart | 37°C | -20°C | GAAGA(8/7) | 5 | 1 | dam | |||
| MlyI | Y | CutSmart | 37°C | -20°C | GAGTC(5/5) | 5 | 0 | |||||
| MmeI | |
Y | CutSmart | 37°C | -20°C | TCCRAC(20/18) | 6 | 2 | CpG; SAM required | IIC | ||
| MnlI | Y | CutSmart® | 37°C | -20°C | CCTC(7/6) | 4 | 1 | |||||
| NmeAIII | |
Y | CutSmart | 37°C | -20°C | GCCGAG(21/19) | 6 | 2 | SAM required | IIC | ||
| PleI | |
Y | CutSmart | 37°C | -20°C | GAGTC(4/5) | 5 | 1 | CpG | |||
| SapI * | Y | CutSmart | 37°C | -20°C | GCTCTTC(1/4) | 7 | 3 | BspQI | IIT | |||
| SfaNI | Y | NEBuffer 3.1 | 37°C | -20°C | GCATC(5/9) | 5 | 4 | CpG |
* Cited for use in Golden Gate Assembly according to current literature
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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