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Home  >Technical Resource  > Restriction Endonucleases > Compatible Cohesive Overhangs

Compatible Cohesive Overhangs

New restriction sites can be generated by ligation of DNA fragments with compatible ends. These ends may be generated by:

1. Cleavage followed by fill-in of 5´ overhangs to generate blunt ends.
2. Cleavage with two restriction endonucleases that produce blunt ends.
3. Cleavage with two restriction endonucleases that produce compatible overhangs.

Compatible ends, generated by each of the above methods, can be ligated to produce DNA sequences that often contain useful restriction endonuclease sites. Generation of these sites is detailed in the following tables.

Restriction endonucleases that produce compatible cohesive ends often produce recleavable ligation products. The combinations listed were identified by computer analysis, and although we have tried to ensure their accuracy, they have not necessarily been confirmed by experimentation.

Where isoschizomers exist, only one member of each set is listed. Only enzymes available from New England Biolabs have been listed.

Enzymes that have degenerate recognition sequences (i.e. recognize more than one sequence) are followed by a specific sequence in parentheses and are only listed if a non-degenerate equivalent does not exist. Be aware that these degenerate enzymes will cleave sequences in addition to the one listed.

A "--" denotes a ligation product that cannot be recleaved.

|  A  |  B  |  C  |  D  |  E  |  H  |  K  |  M  |  N  |  P  |  R  |  S  |  T  |  X  |        single letter code

Enzyme Ligated To Recleaved By
Acc65 I
(G/GTACC)		 Ban I (G/GTACC) Acc65 I, Ban I, Kpn I, Nla IV, Rsa I 
BsiW I, BsrG I Rsa I
Acc I
(GT/CGAC) Aci I, Acl I, BsaH I (GR/CGYC), HinP1 I, Hpa II, Nar I --
(GT/CGAC) Cla I, BstB I, Taq I Taq I
Aci I
(C/CGC) 		Acc I (GT/CGAC), Acl I, Cla I, BstB I, Taq I --
BsaH I (GR/CGCC), HinP1 I, Nar I Aci I
Hpa II Hpa II
Acl I
(AA/CGTT)		 Acc I (GT/CGAC), Aci I, Cla I, BstB I, HinP1 I, Hpa II, Nar I, Taq I --
Age I
(A/CCGGT)		 Ava I (C/CCGGG), Xma I Hpa II, Nci I, ScrF I
BsaW I, BspE I BsaW I, Hpa II
BsrF I (A/CCGGT), SgrA I (CA/CCGGTG) Age I, BsaW I, BsrF I, Hpa II
NgoM IV BsrF I, Hpa II
Apa I
(GGGCC/C)		 Ban II (GGGCC/C), Bsp1286 I (GGGCC/C) Apa I, Ban II, Bsp120 I, Bsp1286 I, Hae III, Nla IV, Sau96 I
ApaL I
(G/TGCAC)		 Sfc I (C/TGCAG) Bsg I
Apo I
(A/AATTY) EcoR I Apo I, Tsp509 I
(G/AATTY) EcoR I Apo I, EcoR I, Tsp509 I
(R/AATTY) Mfe I, Tsp509 I Tsp509 I
Asc I
(GG/CGCGCC)		 Afl III (A/CGCGT), Mlu I BstU I, Hha I
BssH II BssH II, BstU I, Cac8 I, Hha I
Ase I
(AT/TAAT)		 Bfa I, Csp6 I, Nde I --
Mse I Mse I
AsiS I
(GCGAT/CGC) 		BsiE I (CGAT/CG) Dpn II, Pvu I
Pac I Mse I
Pvu I Dpn II, Pvu I
Ava I
(C/CCGGG) Age I, BsaW I, BspE I, BsrF I (R/CCGGY), NgoM IV, SgrA I (CR/CCGGYG) Hpa II, Nci I, ScrF I
(C/TCGAG) Xho I Ava I, Taq I, Xho I
(C/TCGAG) Sal I Taq I
(C/CCGGG) Xma I Ava I, BsaJ I, Hpa II, Nci I, ScrF I, Sma I
Ava II
(G/GWCC)		 PpuM I (RG/GACCY) Ava II, Nla IV, Sau96 I
Rsr II Ava II, Sau96 I
PpuM I (RG/GTCCY) Ava II, BsmF I, Nla IV, Sau96 I
Avr II
(C/CTAGG)		 Nhe I, Spe I, Xba I Bfa I
Sty I (C/CTAGG) Avr II, Bfa I, BsaJ I, Sty I
BamH I
(G/GATCC)		 Bcl I, Dpn II Alw I, Dpn II
Bgl II, BstY I (R/GATCY) Alw I, BstY I, Dpn II
BstY I (G/GATCC) Alw I, BamH I, BstY I, Dpn II, Nla IV
Ban I
(G/GTACC) Acc65 I Acc65 I, Ban I, Kpn I, Nla IV, Rsa I
(G/GCGCC) Kas I Ban I, BsaH I, Hae II, Hha I, Kas I, Nar I, Nla IV
(G/GTACC) BsiW I, BsrG I Rsa I
Ban II
(GGGCC/C) Apa I, Bsp1286 I (GGGCC/C) Apa I, Ban II, Bsp1286 I, Hae III, Nla IV, Sau96 I
(GAGCT/C) Bsp1286 I (GAGCT/C), Sac I Alu I, Ban II, BsiHKA I, Bsp1286 I, Sac I
Bcl I
(T/GATCA) 		BamH I, BstY I (R/GATCY) Alw I, Dpn II
Bgl II, Mbo II Dpn II
Bfa I
(C/TAG)		 Ase I, Csp6 I, Mse I, Nde I --
Bgl II
(A/GATCT)		 BamH I, BstY I (R/GATCY) Alw I, BstY I, Dpn II
Bcl I, Dpn II Dpn II
BsaH I
(GR/CGYC) Acc I (GT/CGAC), Cla I, BstB I, Taq I --
(GA/CGYC) Aci I, HinP1 I Hga I
(GG/CGYC) Aci I, HinP1 I Hha I
(GG/CGYC) Hpa II Aci I
(GA/CGYC) Nar I BsaH I, Hga I
(GG/CGYC) Nar I Ban I, BsaH I, Hae II, Hha I, Nar I, Nla IV
BsaW I
(W/CCGGW)		 Age I, BsrF I (R/CCGGY), SgrA I (CR/CCGGYG) Age I, BsaW I, BsrF I, Hpa II
Ava I (C/CCGGG), Xma I Hpa II, Nci I, ScrF I
BspE I BsaW I, BspE I, Hpa II
BsrF I (R/CCGGY), NgoM IV BsrF I, Hpa II
NgoM IV Hpa II
BsiE I
(CGAT/CG) Pac I Mse I
(CGAT/CG) Pvu I BsiE I, Dpn II, Pvu I
(CGGC/CG) Sac II Aci I
BsiHKA I
(GTGCA/C) 		Bsp1286 I (GTGCA/C) BsiHKA I, Bsp1286 I
Bsp1286 I (GAGCA/C) BsiHKA I, Bsp1286 I
Bsp1286 I (GAGCT/C), Sac I Alu I, Ban II, BsiHKA I, Bsp1286 I, Sac I
Nsi I --
Pst I, Sbf I Bsg I
BsiW I
(C/GTACG)		 Acc65 I, Ban I (G/GTACC), BsrG I Rsa I
Bsp1286 I
(GGGCC/C) Apa I, Ban II (GGGCC/C) Apa I, Ban II, Bsp1286 I, Hae III, Nla IV, Sau96 I
(GTGCA/C) BsiHKA I ApaL I, BsiHKA I, Bsp1286 I
(GGGCC/C) Ban II (GGGCC/C) Ban II, Bsp1286 I
(GAGCT/C) Ban II (GAGCT/C), BsiHKA I, Sac I Alu I, Ban II, BsiHKA I, Bsp1286 I, Sac I
(GWGCW/C) BsiHKA I BsiHKA I, Bsp1286 I
(GTGCA/C) Nsi I --
(GTGCA/C) Pst I, Sbf I Bsg I
BspE I
(T/CCGGA)		 Age I, BsaW I, BsrF I (R/CCGGY), SgrA I (CR/CCGGYG) BsaW I, Hpa II
Ava I (C/CCGGG), Xma I Hpa II, Nci I, ScrF I
BsaW I BsaW I, BspE I, Hpa II
BsrF I (R/CCGGY), NgoM IV Hpa II
BspH I
(T/CATGA)		 Fat I, Nco I, Pci I Fat I, Nla III
BsrF I
(A/CCGGY) Age I, BsaW I Age I, BsaW I, BsrF I, Hpa II
(G/CCGGY) Age I, BsaW I, NgoM IV BsrF I, Hpa II
(R/CCGGY) Ava I (C/CCGGG), Xma I Hpa II, Nci I, ScrF I
(A/CCGGY) BsaW I, BspE I BsaW I, Hpa II
(R/CCGGY) BsaW I, BspE I Hpa II
(G/CCGGY) NgoM IV BsrF I, Cac8 I, Hpa II, Nae I
(CR/CCGGYG) SgrA I BsrF I, Hpa II
BsrG I
(T/GTACA)		 Acc65 I, Ban I (G/GTACC), BsiW I Rsa I
BssH II
(G/CGCGC)		 Afl III (A/CGCGT), Mlu I BstU I, Hha I
Asc I BssH II, BstU I, Cac8 I, Hha I
BstB I
(TT/CGAA)		 Acc I (GT/CGAC), Cla I, Taq I Taq I
Aci I, Acl I, BsaH I (GR/CGYC), HinP1 I, Hpa II, Nar I --
BstY I
(A/GATCY) BamH I, Bgl II Alw I, BstY I, Dpn II
(G/GATCY) BamH I Alw I, BamH I, BstY I, Dpn II, Nla IV
(R/GATCY) Bcl I, Dpn II Dpn II
(G/GATCY) Bcl I, Dpn II Alw I, Dpn II
(A/GATCY) Bgl II Bgl II, BstY I, Dpn II
Cla I
(AT/CGAT)		 Acc I (GT/CGAC), BstB I, Taq I Taq I
Aci I, Acl I, BsaH I (GR/CGYC), HinP1 I, Hpa II, Nar I --
Dpn II/Mbo I/
Sau3A I (/GATC)		 BamH I, BstY I (R/GATCC) Alw I, Dpn II
Bcl I, Bgl II, BstY I (R/GATCY) Dpn II
Eae I
(Y/GGCCR) PspOM I Hae III, Sau96 I
(C/GGCCR) Eag I BsiE I, Eae I, Eag I, Hae III
(T/GGCCR) Eag I Eae I, Hae III
(C/GGCCR) Not I Aci I, BsiE I, Eae I, Eag I, Fnu4H I, Hae III
(T/GGCCR) Not I Aci I, Eae I, Fnu4H I, Hae III
Eag I
(C/GGCCG)		 PspOM I Hae III, Sau96 I
Eae I (Y/GGCCR) Eae I, Hae III
Eae I (C/GGCCG) BsiE I, Eae I, Eag I, Hae III
Not I Aci I, BsiE I, Eae I, Eag I, Fnu4H I, Hae III
EcoR I
(G/AATTC)		 Apo I (G/AATTC) Apo I, EcoR I, Tsp509 I
Apo I (R/AATTY) Apo I, Tsp509 I
Mfe I, Tsp509 I Tsp509 I
Fat I
(/CATG)		 BspH I, Nco I, Pci I Fat I, Nla III
HinP1 I
(G/CGC)		 Acc I (GT/CGAC), Acl I, Cla I, BstB I, Taq I --
Aci I, BsaH I (GR/CGCC), Nar I Hha I
BsaH I (GR/CGTC) Hga I
Hpa II Aci I
Hpa II/Msp I
(C/CGG)		 Acc I (GT/CGAC), Acl I, Cla I, BstB I, Taq I --
Aci I, BsaH I (GR/CGCC), HinP1 I, Nar I Aci I
Kas I
(G/GCGCC)		 Ban I (G/GCGCC) Ban I, BsaH I, Hae II, Hha I, Kas I, Nar I, Nla IV
Mfe I
(C/AATTG)		 Apo I (R/ATTTY), EcoR I, Tsp509 I Tsp509 I
Mlu I
(A/CGCGT)		 Afl III (A/CGCGT) Afl III, BstU I, Mlu I
Asc I, BssH II BstU I, Hha I
Mse I
(T/TAA)		 Ase I Mse I
Bfa I, Csp6 I, Nde I --
Nar I
(GG/CGCC)		 Acc I (GT/CGAC), Acl I, Cla I, BstB I, Taq I --
Aci I, HinP1 I Hha I
BsaH I (GR/CGCC) Ban I, BsaH I, Hae II, Hha I, Nar I, Nla IV
BsaH I (GR/CGTC) BsaH I, Hga I
Hpa II Aci I
Nco I
(C/CATGG)		 BspH I, Fat I, Pci I Fat I, Nla III
Nde I
(CA/TATG)		 Ase I, Bfa I, Csp6 I, Mse I --
NgoM IV
(G/CCGGC)		 Age I, BsaW I, BsrF I (R/CCGGY), SgrA I BsrF I, Hpa II
Ava I (C/CCGGG), Xma I Hpa II, Nci I, ScrF I
BsaW I, BspE I Hpa II
BsrF I (R/CCGGC), SgrA I BsrF I, Cac8 I, Hpa II, Nae I
Nhe I
(G/CTAGC)		 Avr II, Spe I, Sty I (C/CTAGG), Xba I Bfa I
Nla III
(CATG/)		 Sph I, Nsp I Nla III
Not I
(GC/GGCCGC)		 PspOM I Aci I, Eae I, Fnu4H I, Hae III
Eag I Aci I, BsiE I, Eae I, Eag I, Fnu4H I, Hae III
Eae I (Y/GGCCR) Aci I, BsiE I, Eae I, Fnu4H I, Hae III
Nsi I
(ATGCA/T)		 BsiHKA I (GTGCA/C), Bsp1286 I (GTGCA/C), Pst I, Sbf I --
Nsp I
(RCATG/Y)		 Nla III, Sph I Nla III
Pac I
(TTAAT/TAA)		 BsiE I (CGAT/CG), Pvu I Mse I
Pci I
(A/CATGT)		 BspH I, Fat I, Nco I Fat I, Nla III
PpuM I
(RG/GWCCY) Ava II, Rsr II Ava II, Sau96 I
(GG/GTCCY) Ava II, Rsr II Ava II, BsmF I, Nla IV, Sau96 I
(GG/GACCY) Ava II, Rsr II Ava II, Nla IV, Sau96 I
PspOM I
(G/GGCCC)		 Eae I (Y/GGCCR), Eag I Hae III, Sau96 I
Not I Aci I, Fnu4H I, Hae III, Sau96 I
PspX I
(VC/TCGAGB)		 Xho I, Tli I Xho I, Tli I
Sal I Taq I
Pst I
(CTGCA/G)		 BsiHKA I, Bsp1286 I (GTGCA/C) Bsg I
Nsi I --
Sbf I Pst I
Pvu I
(CGAT/CG)		 AsiS I Dpn I, Pvu I
Pac I Mse I
BsiE I (CGAT/CG) BsiE I, Dpn II, Pvu I
Rsr II
(CG/GWCCG)		 Ava II, PpuM I (RG/GACCY) Ava II, Sau96 I
PpuM I (RG/GACCY) Ava II, Nla IV, Sau96 I
PpuM I (RG/GTCCY) Ava II, BsmF I, Nla IV, Sau96 I
Sac I
(GAGCT/C)		 Ban II (GAGCT/C), BsiHKA I, Bsp1286 I (GAGCT/C) Alu I, Ban II, BsiHKA I, Bsp1286 I, Sac I
Sac II
(CCGC/GG)		 BsiE I (CGGC/CG) Aci I
Sal I
(G/TCGAC)		 PspX I, Xho I Taq I
Sbf I
(CCTGCA/GG)		 BsiHKA I, Bsp1286 I (GTGCA/C) Bsg I
Nsi I --
Pst I Pst I
Sfc I
(C/TGCAG)		 ApaL I Bsg I
SgrA I
(CR/CCGGYG)		 See BsrF I  
Spe I
(A/CTAGT)		 Avr II, Nhe I, Sty I (C/CTAGG), Xba I Bfa I
Sph I
(GCATG/C)		 Nla III, Nsp I Nla III
Sty I Avr II Avr II, Bfa I, BsaJ I, Sty I
(C/CTAGG) Nhe I, Spe I, Xba I Bfa I
(C/CATGG) BspH I Nla III
Nco I BsaJ I, Nco I, Nla III, Sty I
Taq I
(T/CGA)		 Acc I (GT/CGAC), Cla I, BstB I Taq I
Aci I, Acl I, BsaH I (GR/CGYC), HinP1 I,Hpa II, Nar I --
Tsp509 I
(/AATT)		 Apo I (R/AATTY), EcoR I, Mfe I Tsp509 I
Xba I
(T/CTAGA)		 Avr II, Nhe I, Spe I, Sty I (C/CTAGG) Bfa I
Xho I/Tli I
(C/TCGAG 		PspX I Xho I, Tli I
Sal I Taq I
Xma I
(C/CCGGG		 Age I, BsaW I, BspE I, BsrF I, NgoM IV, SgrA I Hpa II, Nci I, ScrF I
Ava I (C/CCGGG) Ava I, BsaJ I, Hpa II, Nci I, ScrF I, Sma I, Xma I