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®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
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		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	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		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-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; 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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