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 45 Type IIS restriction enzymes. This table allows you to sort our enzymes by feature for easy comparison.
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     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     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 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
  IIC
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
  IIC
BsmAI
N CutSmart 55°C 50% -20°C GTCTC(1/5) 5 4 BcoDI CpG
BsmBI-v2 *
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
  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 NEBuffer 2.1
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-v2
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     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-v2 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 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
  IIC
PaqCI
Y CutSmart 37°C
-20°C CACCTGC(4/8) 7 4   CpG
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
2+site 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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