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
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.

This content is covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at gbd@neb.com. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.

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