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Recleavable Filled-in 5´ Overhangs

New England Biolabs Technical Literature - Updated

Recleavable Filled in 5´ -Overhangs

The table below lists restriction endonucleases that generate 5´ overhangs which, if filled-in and ligated, result in new restriction sites. 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.

Restriction enzymes which have degenerate recognition specificities (i.e. recognize more than one sequence) have been excluded from this list. Where isoschizomers exist, only one member of each set is listed. Only commercially available enzymes have been listed.

Example:	EcoR I Fragments			XmnI and AseI Sites
	5´ ...G	AATTC...3´	Fill-in and Ligate-->	5´ ...GAATTAATTC...3´
	3´ ...CTTAA	G...5´	Fill-in and Ligate-->	3´ ...CTTAATTAAG...5´

Table Notes:
Enzymes in red have recognition sequences of 6 or 8 bases. Sequence length is indicated by superscript (e.g., Asc I⁸ = 8-base cutter).

Enzymes in parenthesis indicate that the new sequence is still a substrate for the original enzyme.

A superscript 2 indicates that two identical sites have been generated within the filled-in/ligated sequence. For example, fill-in/ligation of Afl II generates the sequence CTTAATTAAG which contains two Mse I sites (TTAA).

| A | B | C | D | E | H | K | M | N | P | S | T | X |

Enzyme	Cleavage Site	After Fill-in/Ligation	Recleaved By
Acc65 I	G/GTACC	GGTACGTACC	BsaA I, HpyCH4 IV, Rsa I, SnaB I⁶
Aci I	C/CGC	CCGCGC	(Aci I), BstU I, Hha I
Acl I	AA/CGTT	AACGCGTT	Afl III, BstU I, Mlu I⁶
Afl II	C/TTAAG	CTTAATTAAG	Mse I², Pac I⁸, Tsp509 I
Age I	A/CCGGT	ACCGGCCGGT	BsiE I, BsrF I, Eae I, Eag I⁶, Hae III, Hpa II
ApaL I	G/TGCAC	GTGCATGCAC	Cac8 I, Nla III, Nsp I, Sph I⁶
Asc I	GG/CGCGCC	GGCGCGCGCGCC	BssH II, BstU I, Cac8 I, Hha I
Avr II	C/CTAGG	CCTAGCTAGG	Alu I, Bfa I
BamH I	G/GATCC	GGATCGATCC	Alw I, Cla I⁶, Dpn II, Taq I
Bcl I	T/GATCA	TGATCGATCA	Cla I⁶, Dpn II, Taq I
Bfa I	C/TAG	CTATAG	Sfc I
Bgl II	A/GATCT	AGATCGATCT	Cla I⁶, Dpn II², Taq I
BsiW I	C/GTACG	CGTACGTACG	BsaA I, (BsiW I), HpyCH4 IV, Rsa I, SnaB I⁶
BspD I/Cla I	AT/CGAT	ATCGCGAT	BstU I, Nru I⁶
BspE I	T/CCGGA	TCCGGCCGGA	BsiE I, Eae I, Eag I⁶, Hae III, Hpa II
BspH I	T/CATGA	TCATGCATGA	Nla III, Nsi I⁶
BsrG I	T/GTACA	TGTACGTACA	BsaA I, Rsa I, SnaB I⁶
BssH II	G/CGCGC	GCGCGCGCGC	(BssH II), BstU I, Cac8 I, Hha I
BstB I	TT/CGAA	TTCGCGAA	BstU I, Nru I⁶
Dpn II/Mbo I/Sau3A I	/GATC	GATCGATC	Cla I⁶, (Dpn II), Taq I
Eag I	C/GGCCG	CGGCCGGCCG	BsiE I, BsrF I, Cac8 I, Eae I², (Eag I²), Fse I⁸, Hae III, Hpa II, Nae I
EcoR I	G/AATTC	GAATTAATTC	Ase I⁶, Mse I, Tsp509 I, Xmn I⁶
HinP1 I	G/CGC	GCGCGC	BssH II⁶, BstU I, Cac8 I, (Hha I)
Hind III	A/AGCTT	AAGCTAGCTT	Alu I, Bfa I, Cac8 I, Nhe I⁶
Hpa II/Msp I	C/CGG	CCGCGG	Aci I, BsaJ I, BstU I, Btg I, MspA1 I, Sac II⁶
HpyCH4 IV	A/CGT	ACGCGT	Afl III, BstU I, Mlu I⁶
Kas I	G/GCGCC	GGCGCGCGCC	BssH II⁶, BstU I, Cac8 I, Hha I
Mfe I	C/AATTG	CAATTAATTG	Ase I⁶, Tsp509 I²
Mlu I	A/CGCGT	ACGCGCGCGT	BssH II⁶, BstU I, Cac8 I, Hha I
Nar I	GG/CGCC	GGCGCGCC	Asc I⁸, BssH II, BstU I, Cac8 I, Hha I
Nco I	C/CATGG	CCATGCATGG	Nla III, Nsi I⁶
NgoM IV	G/CCGGC	GCCGGCCGGC	BsiE I, BsrF I, Cac8 I, Eae I, Eag I⁶, Hae III, Hpa II, (NgoM IV²)
Nhe I	G/CTAGC	GCTAGCTAGC	Alu I, Bfa I, Cac8 I, (Nhe I)
Not I	GC/GGCCGC	GCGGCCGGCCGC	Aci I, BsiE I, BsrF I, Cac8 I, Eae I, Eag I⁶, Fnu4H I, Fse I⁸, Hae III, Hpa II, Nae I⁶
PaeR7 I/Xho I	C/TCGAG	CTCGATCGAG	BsiE I, Dpn II, Pvu I⁶, (Taq I²)
Pci I	A/CATGT	ACATGCATGT	HpyCH4 V, (Nla III²⁾, Nsi I⁶
PspOM I	G/GGCCC	GGGCCGGCCC	BsrF I, Cac8 I, Fse I⁶, Hae III, Hpa II, Nae I, Sau96 I
Sal I	G/TCGAC	GTCGATCGAC	BsiE I, Dpn II, Pvu I⁶, Taq I
Spe I	A/CTAGT	ACTAGCTAGT	Alu I, Bfa I
Taq I	T/CGA	TCGCGA	BstU I, Nru I⁶
Tsp509 I	/AATT	AATTAATT	Ase I⁶, Mse I, (Tsp509 I²)
Xba I	T/CTAGA	TCTAGCTAGA	Alu I, Bfa I
Xma I	C/CCGGG	CCCGGCCGGG	BsiE I, Eae I, Eag I⁶, Hae III, Hpa II, Nci I, ScrF I