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Promega Corporation

Restriction Enzyme Reference Information

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Relative Activity of Restriction Enzymes in Promega 10X Buffers

The 10X Reaction Buffer supplied with each restriction enzyme is optimized to give 100% activity. In many cases, good activity is also obtained using one of the 4-CORE® 10X Buffers (Cat.# R9921). Table 3.1 may be used to select the best buffer for digestions with multiple restriction enzymes. Enzyme activity is expressed as a percent of the activity obtained with the optimized buffer for each enzyme.

Promega Enzyme Buffer Supplied with Enzyme 4 CORE® Buffers MULTI-CORE Buffer Enzyme
Assay
Temperature
A B C D
AatII J 50-75% 10-25% <10% <10% <10% 37°C
AccI G 50-75% 25-50% 25-50% 10-25% 25-50% 37°C
AccIII F <10% 10-25% 25-50% 25-50% <10% 65°C
Acc65I D 10-25% 50-75% 75-100% 100% 100% 37°C
AccB7I E 10-25% 50-75% 100%** <10% 100% 37°C
AgeI K 25-50% 25-50% 25-50% 50-75% 100% 37°C
AluI B 75-100% 100% 75-100% 10-25% 10-25% 37°C
Alw26I C 10-25% 25-50% 100% 10-25% 75-100% 37°C
Alw44I C <10% 25-50% 100% 25-50% 100% 37°C
ApaI A 100% 50-75% 50-75% <10% 75-100% 37°C
AvaI B 10-25% 100% 50-75% 25-50% <10% 37°C
AvaII C 50-75% 50-75% 100% 25-50% 25-50% 37°C
Bal I G 10-25% <10% <10% <10% <10% 37°C
BamHI E 75-100% ** 75-100% 75-100% 50-75% 75-100% 37°C
BanI G 25-50% 25-50% 10-25% <10% 100% 50°C
BanII E 75-100% 75-100% 75-100% 25-50% 100% 37°C
BbuI A 100% 75-100% 75-100% <10% 100% 37°C
Bcl I C 10-25% 75-100% 100% 50-75% 10-25% 50°C
Bgl I D 10-25% 25-50% 75-100% 100% 100% 37°C
Bgl II D 25-50% 75-100% 75-100% 100% <10% 37°C
BsaMI D 10-25% 25-50% 50-75% 100% 25-50% 65°C
BsaOI C 10-25% 50-75% 100% 25-50% 100% 50°C
Bsp I286I A 100% 50-75% 25-50% 10-25% 75-100% 37°C
BsrBRI H 10-25% 50-75%** 100%** 50-75% 100% 65°C
BsrSI D 10-25% 25-50% 10-25% 100% 100% 65°C
BssH II H 75-100% 50-75% 75-100% 50-75% 75-100% 50°C
Bst71 I D 10-25% 25-50% 25-50% 100% 10-25% 50°C
Bst98I D <10% 10-25% 10-25% 100% 25-50% 37°C
BstEII D 25-50% 50-75% 50-75% 100% 100% 60°C
BstOI C 10-25% 25-50% 100% 25-50% <10% 60°C
BstXI D <10% 10-25% 25-50% 100% 10-25% 50°C
BstZI D <10% <10% 10-25% 100% 10-25% 50°C
Bsu36I E <10% 25-50% 50-75% 25-50% 50-75% 37°C
CfoI B 75-100% 100% 75-100% 25-50% 100% 37°C
ClaI C 75-100% 75-100% 100% 75-100% 100% 37°C
CspI K <10% 10-25% 25-50% 50-75% 10-25% 30°C
Csp45I B 25-50% 100% 50-75% 25-50% 50-75% 37°C
DdeI D 25-50% 25-50% 50-75% 100% 25-50% 37°C
DpnI B 50-75% 100% 75-100% 50-75% 100% 37°C
DraI B 75-100% 100% 75-100% 50-75% 25-50% 37°C
EclHKI E <10% <10% 75-100% 10-25% 50-75% 37°C
Eco47III D <10% 25-50% 50-75% 100% 25-50% 37°C
Eco52I L <10% <10% 10-25% 25-50% <10% 37°C
EcoICRI B 10-25% 100% 75-100% <10% 100% 37°C
EcoRI H 25-50% 50-75% 50-75% 50-75% 100% 37°C
EcoRV D 10-25% 25-50% 50-75% 100% 100% 37°C
FokI B 75-100% 100% 75-100% 25-50% 50-75% 37°C
HaeII B 50-75% 100% 50-75% 10-25% 100% 37°C
HaeIII C 75-100% 75-100% 100% 50-75% 100% 37°C
HhaI C 50-75% 75-100% 100% 50-75% 75-100% 37°C
HincII B 25-50% 100% 25-50% 50-75% 100% 37°C
HindIII E 25-50% 100% 75-100% 10-25% 50-75% 37°C
HinfI B 50-75% 100% 75-100% 75-100% 50-75% 37°C
HpaI J 25-50% 50-75% 25-50% 10-25% 100% 37°C
HpaII A 100% 50-75% 50-75% 10-25% 100% 37°C
Hsp92I F 10-25% 75-100% 50-75% 25-50% 10-25% 37°C
Hsp92II K 10-25% 25-50% 25-50% <10% <10% 37°C
I-PpoI I-Ppo I 10-25% 25-50% 25-50% 25-50% - 37°C
KpnI J 100%** 25-50% 25-50% <10% 75-100% 37°C
MboI C 10-25% 75-100% 100% 50-75% <10% 37°C
MboII B 10-25% 100% 50-75% 75-100% 100% 37°C
MluI D 10-25% 25-50% 50-75% 100% 10-25% 37°C
MspI B 75-100% 100% 75-100% 25-50% 25-50% 37°C
MspA1I C 25-50% 100%** 100% 10-25% 100% 37°C
NaeI A 100% 50-75% 25-50% <10% 50-75% 37°C
NarI G 75-100% 50-75% 75-100% 25-50% 50-75% 37°C
NciI B 100%** 100% 25-50% 25-50% 50-75% 37°C
NcoI D 50-75% 75-100% 75-100% 100% 75-100% 37°C
NdeI D <10% <10% 25-50% 100% 25-50% 37°C
Nde II D <10% <10% 10-25% 100% 25-50% 37°C
NgoMIV MULTI-
CORE
100%** 100%** 100%** <10% 100% 37°C
NheI B 75-100% 100% 75-100% 10-25% 100% 37°C
Not I D <10% 10-25% 25-50% 100% 25-50% 37°C
NruI K <10% <10% <10% 50-75% 10-25% 37°C
NsiI D 10-25% 50-75% 50-75% 100% 10-25% 37°C
PstI H 10-25% 50-75% 50-75% 50-75% 25-50% 37°C
PvuI D 10-25% 25-50% 50-75% 100% <10% 37°C
PvuII B 25-50% 100% 50-75% 25-50% 50-75% 37°C
RsaI C 75-100% 75-100% 100% <10% <10% 37°C
SacI J 75-100% 25-50% 25-50% <10% 100% 37°C
SacII C 100%* 50-75% 100% 50-75% <10% 37°C
Sal I D <10% 10-25% 25-50% 100% <10% 37°C
Sau3AI B 25-50% 100% 75-100% <10% 100% 37°C
Sau96I C 25-50% 25-50% 100% 50-75% 50-75% 37°C
ScaI K <10% 100%** 50-75% 75-100% 10-25% 37°C
SfiI B 75-100% 100% 75-100% 25-50% 75-100% 50°C
SgfI (a) C 25-50% 25-50% 100% <10% <10% 37°C
SinI A 100% 75-100% 50-75% 10-25% 100% 37°C
SmaI J <10% <10% 10% <10% 100% 25°C
SnaBI B 50-75% 100% 50-75% <10% 100% 37°C
SpeI B 75-100% 100% 75-100% 75-100% 100% 37°C
SphI K 75-100% 75-100%** 100% 75-100% 10-25% 37°C
SspI E 10-25% 50-75% 50-75% 75-100% 50-75% 37°C
StuI B 75-100% 100% 75-100% 50-75% 50-75% 37°C
StyI F 25-50% 75-100% 75-100% 75-100% <10% 37°C
TaqI E 10-25% 25-50% 50-75% 50-75% 100% 65°C
Tru9I F 75-100% 50-75% 75-100% 25-50% 25-50% 65°C
Tth111 I B 50-75% 100% 75-100% 25-50% 100% 65°C
VspI D <10% 25-50% 75-100% 100% <10% 37°C
XbaI D 50-75% 75-100% 75-100% 100% 100% 37°C
XhoI D 25-50% 75-100% 75-100% 100% 10-25% 37°C
XhoII C 25-50% 25-50% 100% 10-25% <10% 37°C
XmaI B 50-75% 100% 25-50% <10% 50-75% 37°C
XmnI B 75-100% 100% 75-100% 10-25% 75-100% 37°C

*Sac II exhibits 100% activity with linear substrates in buffer A. It exhibits very poor activity with supercoiled substrates in this buffer.
**These buffers are not recommended due to star activity.

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Activity of Cloning Enzymes in Buffer H

EcoR I and PstI are commonly used enzymes in double digests and cloning applications. Their optimal buffer is H. Table 3.2 provides the relative activity of other cloning enzymes in Buffer H for double digest purposes.

AatII <10%   MluI 100-125%*
AccI <10%   NcoI 100-125%*
Acc65I 100-125%*   NheI 10-25%
ApaI <10%   NotI  100-125%*
AvaI 10-25%   NsiI >125%*
BamHI 50-75%   PstI 100%
BbuI 10-25%   SacI 25-50%
Bcl I 50-75%   SacII >125%*
Bgl I 75-100%   Sal I 25-50%
BstXI 75-100%   SfiI 50-75%
BstZI 75-100%   SmaI <10%
ClaI 50-75%   SpeI 25-50%
CspI 100-125%*   SphI >125%*
Csp45I 25-50%   SpoI <10%
Eco52I 50-75%   SspI 100-125%*
EcoRI 100%   StyI 50-75%
EcoRV 50-75%   XbaI 100-125%*
HincII 50-75%   XhoI 100-125%*
HindIII 25-50%   XmaI <10%
KpnI <10%      

*Unit activity is based on recommended buffer. In buffer H, some enzymes have enhanced activity.

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Composition of Promega Restriction Enzyme Reaction Buffers 1X

Promega Restriction Enzyme Reaction Buffers are provided as 10X stock solutions. Table 3.3 provides compositions for Restriction Enzyme Reaction Buffers, listed as 1X concentrations.

Buffer pH
(at 37°C)
Tris-HCl
(mM)
MgCl2
(mM)
NaCl
(mM)
KCl
(mM)
DTT
(mM)
A 7.5 6 6 6 1
B 7.5 6 6 50 1
C 7.9 10 10 50 1
D 7.9 6 6 150 1
E 7.5 6 6 100 1
F 8.5 10 10 100 1
G 8.2 50 5
H 7.5 90 10 50
J 7.5 10 7 50 1
K 7.4 10 10 150
L 9.0 10 3 100

MULTI-CORE™ Buffer (1X) = 25mM Tris-Acetate, pH 7.5 (at 37°C), 100mM potassium acetate, 10mM magnesium acetate, 1mM DTT.

Notes:

  1. For each 10°C rise in temperature between 0°C and 25°C, the pH of Tris buffers decreases 0.31 pH units.
  2. For each 10°C rise in temperature between 25°C and 37°C, the pH of Tris buffers decreases 0.25 pH units.

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Heat Inactivation of Restriction Enzymes

Heat-inactivation of restriction enzymes may be performed when a subsequent reaction can be performed in the same reaction buffer, or when the reaction will be diluted for the next application. This will eliminate the need for extra ethanol precipitations or clean-up steps. Table 3.4 lists the sensitivity of Promega's restriction enzymes to heat-inactivation.

Promega
Enzyme
Heat
Inactivated
AatII +
AccI -
AccIII -
Acc65I +
AccB7I +
AgeI +
AluI +
Alw26I +
Alw44I +
ApaI +
AvaI +/-
AvaII +
Bal I +
BamHI +
BanI -
BanII +
BbuI +
Bcl I -
Bgl I +
Bgl II -
BsaMI -
BsaOI -
Bsp1286I +
BsrBRI -
BsrS I -
BssHII -
Bst71I -
Bst98I -
BstEII -
BstOI -
BstXI +/-
BstZI -
Bsu36I -
CfoI +/-
ClaI +
CspI +
Csp45I +
DdeI +/-
DpnI +
DraI +
EclHKI +
Eco47III +
Eco52I +
EcoICRI +
EcoRI +
EcoRV +
FokI +
HaeII -
HaeIII -
HhaI +
HincII +
HindIII +
Promega
Enzyme
Heat
Inactivated
HinfI -
HpaI -
HpaII -
Hsp92I +
Hsp92II +
I-Ppo I +
KpnI +/-
MboI +
MboII +
MluI +/-
MspI +
MspA1 I +
NaeI +
NarI +
NciI +
NcoI +
NdeI +
NdeII +
NgoMIIV +
NheI +
NotI +
NruI +
NsiI +/-
PstI +
PvuI -
PvuII +
RsaI +
SacI +
SacII +
Sal I +
Sau3AI +
Sau96I -
ScaI +
SfiI -
SgfI(a) +/-
SinI +
SmaI +
SnaBI -
SpeI +
SphI +
SspI +
StuI +
StyI +
TaqI -
Tru9I -
Tth111 I -
VspI +
XbaI -
XhoI +
XhoII +
XmaI +
XmnI +

Key:

+ greater than 95% inactivation (DNA is undigested).

– less that 95% inactivation (DNA digest is complete, i.e., at least 5% of the initial 20 activity units [at least 1 unit] remain(s).)

+/– partial inactivation (DNA is partially digested).

Conditions: Twenty units of enzyme in 50µl of its optimal buffer were heated at 65°C for 15 minutes. 1µg of DNA was added and incubated for 1 hour in accordance with the unit definition, then analyzed by agarose gel electrophoresis.

(a)U.S. Pat. No. 5,391,487 has been issued to Promega Corporation for Restriction Endonuclease SgfI.

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The Effect of Site-Specific Methylation on Promega Restriction Enzymes

Heat-inactivation of restriction enzymes may be performed when a subsequent reaction can be performed in the same reaction buffer, or when the reaction will be diluted for the next application. This will eliminate the need for extra ethanol precipitations or clean-up steps. Table 3.4 lists the sensitivity of Promega's restriction enzymes to heat-inactivation.

If little or no cutting is seen with a restriction enzyme, one possibility is that DNA methylation (or lack of methylation, in the case of Dpn I) is a problem. The sensitivity of Promega's restriction enzymes to DNA methylation is summarized in Table 3.5. If the enzyme used is sensitive to methylation, check the genetic characteristics of the bacterial strain or expression system from which the DNA was purified. The interfering type of methylation may be present.

Prokaryotic Methylation

  • dcm Cytosine methylase - Methylates the C5 position of the internal cytosine residue in the sequence 5´...CCTGG...3´ and 5´...CCAGG...3´
  • dam Adenine methylase - Methylates the N6 position of the adenine residue in the sequence 5´...GATC...3´

Eukaryotic Methylation

  • CpG Methylates the C5 position of the cytosine residue in the dinucleotide recognition sequence 5´...CG...3´
  • CpNpGp Methylates the C5 position of the cytosine residue in the trinucleotide 5´...CNG...3´ (N = any base)
Enzyme Recognition Sequence dam dcm CpG CpNpG
AatII GACGTC i i s i
AccB7I CCANNNNNTGG i s(ol) i i
AccIII TCCGGA s(ol) i i i
Acc65I GGTACC i s(ol) i i
ApaI GGGCCC i s(ol) s(ol) i
AvaI CYCGRG i i s i
AvaII GGWCC i s(ol) s(ol) s(ol)
Bal I TGGCCA i s(ol) i s(ol)
BamHI GGATCC i i i s(ol)
BanII GRGCYC i i i i
BbuI GCATGC i i i i
Bcl I TGATCA s i i i
BglI GCCNNNNNGGC i i s(ol) s(ol)
BssHII GCGCGC i i s i
BglII AGATCT i i i s(ol)
BsaOI CGRYCG i i n/a n/a
Bsp1286 I GDGCHC i i i i
BsrBRI GATNN/NNATC s i i i
BstEII GGTNACC i i i i
BstOI CCWGG i i i n/a
BstXI CCANNNNNNTGG i i i i
BstZI CGGCCG i i s(ol) s(ol)
CfoI GCGC i i s s(ol)
ClaI ATCGAT s(ol) i s i
CspI CGGWCCG i i s s
Csp45I TTCGAA i i s i
DdeI CTNAG i i i s(ol)
Eco47III AGCGCT i i s i
Eco52I CGGCCG i i s i
EcoRI GAATTC i i s(ol) i
FokI GGATG i i i i
HaeIII GGCC i i i s(ol)
HhaI GCGC i i s s(ol)
HincII GTYRAC i i i i
HindIII AAGCTT i i i i
HpaII CCGG i i s s
KpnI GGTACC i i i i
MboI GATC s i i i
MboII GAAGA(8/7) s(ol) i i i
MluI ACGCGT i i s i
MspI CCGG i i s s
NaeI GCCGGC i i s s
NarI GGCGCC i i s i
NdeII GATC s i i i
NgoMIV GCCGGC i i s s
NheI GCTAGC i i s(ol) s(ol)
NotI GCGGCCGC i i s s
NruI TCGCGA s(ol) i s i
PstI CTGCAG i i i s
PvuI CGATCG i i s s(ol)
PvuII CAGCTG i i i s
SacI GAGCTC i i i n/a
SacII CCGCGG i i s s
Sal I GTCGAC i i s i
Sau3AI GATC i i s(ol) s(ol)
Sau96I GGNCC i s(ol) s(ol) s(ol)
ScaI AGTACT i i i i
SfiI GGCCNNNNNGGCC i s(ol) s(ol) s(ol)
SgfI (a) GCGATCGC i n/a s n/a
SinI GGWCC i i i s(ol)
SmaI CCCGGG i i s s
SnaBI TACGTA i i s i
SphI GCATGC i i i i
StuI AGGCCT i s(ol) i s(ol)
TaqI TCGA s(ol) i i i
Tth111 I GACNNNGTC i i i i
XbaI TCTAGA s(ol) i i i
XhoI CTCGAG i i s i
XhoII RGATCY i i i s(ol)
XmaI CCCGGG i i i n/a
XmnI GAANNNNTTC i i n/a n/a

Key:

s = sensitive to this methylation

i = insensitive to this methylation

s(ol) = overlapping - (sensitive when restriction site overlaps methylation sequence)

n/a = information not available

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Methylation Sensitivity of Isoschizomer Neoschizomer Pairs

Heat-inactivation of restriction enzymes may be performed when a subsequent reaction can be performed in the same reaction buffer, or when the reaction will be diluted for the next application. This will eliminate the need for extra ethanol precipitations or clean-up steps. Table 3.4 lists the sensitivity of Promega restriction enzymes to heat-inactivation.

Isoschizomers and neoschizomers often differ in their sensitivity to methylation. This can be a simple way to differentiate the methylation state of DNA. Iso/neoschizomer pairs sold by Promega are listed below.

Methylated Sequence Cleaved by Not Cleaved by
m4CCGG MspI (C/CGG) HpaII (C/CGG)
Cm5CGG MspI (C/CGG) HpaII (C/CGG)
Cm4CGG MspI (C/CGG) HpaII (C/CGG)
CCm5CGGG XmaI (C/CCGGG) SmaI (CCC/GGG)
Gm6ATC Sau3AI (/GATC) MboI, NdeII (/GATC)
GATm5C MboI, NdeII (/GATC) Sau3AI (/GATC)
GATm4C MboI (/GATC) Sau3AI (/GATC)
GGTACm5C KpnI (GGTAC/C) Acc65I (G/GTACC)

Reference

McClelland, M., Nelson, M. and Raschke, E. (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucl. Acids Res. 22, 3640.

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Recognition Sites in Common DNA Substrates

Industry standards for enzyme unit definitions generally use DNA substrates other than plasmid DNA. It is therefore necessary to determine how many units of enzyme will be needed in cloning experiments involving plasmid vectors. Linear DNA such as lambda can be cleaved more efficiently than closed circular plasmid DNA, and fewer units of enzyme per microgram of DNA may be required for a complete digest. The number of sites per microgram of DNA must also be considered. For example, 1µg of lambda contains 0.0317 picomoles of DNA. For a 3,000bp plasmid there are 0.5pmol DNA in 1µg. Therefore, in 1µg of lambda DNA there will be fewer cut sites to digest than in 1µg of plasmid DNA. Table 3.7 provides information on the number of recognition sites for Promega's restriction enzymes in various DNA substrates.

Enzyme Recognition Site Number of Sites in:
lambda Ad2 phiX174 pUC18 M13mp18 pBR322
AatII GACGT/C 10 3 1 1 0 1
AccI GT/MKAC 9 17 2 1 1 2
AccIII T/CCGGA 24 8 0 0 0 1
Acc65I G/GTACC 2 8 0 1 1 0
AccB7I CCANNNN/NTGG 14 18 2 0 0 2
AgeI A/CCGGT 13 5 0 0 0 0
AluI AG/CT 143 158 24 16 27 17
Alw26I GTCTC(1/5) 37 60 4 4 5 3
Alw44I G/TGCAC 4 7 1 3 0 3
ApaI GGGCC/C 1 12 0 0 0 0
AvaI C/YCGRG 8 40 1 1 2 1
AvaII G/GWCC 35 73 1 2 1 8
Bal I TGG/CCA 18 17 0 0 1 1
BamHI G/GATCC 5 3 0 1 1 1
BanI G/GYRCC 25 57 3 4 7 9
BanII GRGCY/C 7 57 0 1 2 2
BbuI GCATG/C 6 8 0 1 1 1
Bcl I T/GATCA 8 5 0 0 0 0
Bgl I GCCNNNN/NGGC 29 20 0 2 1 3
Bgl II A/GATCT 6 11 0 0 1 0
BsaMI GATTGCN/ 46 10 4 0 1 1
BsaOI CGRY/CG 22 50 1 5 4 7
Bsp1286I GDGCH/C 38 105 3 10 5 10
BsrBRI GATNN/NNATC 21 2 2 0 2 1
BsrSI ACTGGN/ 110 86 9 11 18 19
BssHII G/CGCGC 6 52 1 0 0 0
Bst71I GCAGC(8/12) 199 179 14 12 10 21
Bst98I C/TTAAG 3 4 2 0 0 0
BstEII G/GTNACC 13 10 0 0 0 0
BstOI CC/WGG 71 136 2 5 7 6
BstXI CCANNNNN/NTGG 13 10 3 0 0 0
BstZI C/GGCCG 2 19 0 0 0 1
Bsu36I CC/TNAGG 2 7 0 0 1 0
CfoI GCG/C 215 375 18 17 26 31
ClaI AT/CGAT 15 2 0 0 2 1
CspI CG/GWCCG 5 2 0 0 0 0
Csp45I TT/CGAA 7 1 0 0 0 0
DdeI C/TNAG 104 97 14 6 29 8
DpnI GmeA/TC 116 87 0 15 7 22
DraI TTT/AAA 13 12 2 3 5 3
EclHKI GACNNN/NNGTC 9 9 1 1 0 1
Eco47 III ACG/GCT 2 13 0 0 2 4
Eco52 I C/GGCCG 2 19 0 0 0 1
EcoICRI GAG/CTC 2 16 0 1 1 0
EcoRI G/AATTC 5 5 0 1 1 1
EcoRV GAT/ATC 21 9 0 0 0 1
FokI GGATG(9/13) 149 78 8 5 4 12
HaeII RGCGC/Y 48 76 8 3 6 11
HaeIII GG/CC 149 216 11 11 15 22
HhaI GCG/C 215 375 18 17 26 31
HincII GTY/RAC 35 25 13 1 1 2
HindIII A/AGCTT 7 12 0 1 1 1
HinfI G/ANTC 148 72 21 6 27 10
HpaI GTT/AAC 14 6 3 0 0 0
HpaII C/CGG 328 171 5 13 18 26
Hsp92I GR/YCGC 40 44 7 3 1 6
Hsp92II CATG/ 181 183 22 11 16 26
I-PpoI CTCTCTTAA/GGTAGC 0 0 0 0 0 0
KpnI GGTAC/C 2 8 0 1 1 0
MboI /GATC 116 87 0 15 7 22
MboII GAAGA(8/7) 130 113 11 7 12 11
MluI M/CGCGT 7 5 2 0 0 0
MspI C/CGG 328 171 5 13 18 26
MspAI CMG/CKG 75 95 4 6 4 5
NaeI GCC/GGC 1 13 0 0 1 4
NarI GG/CGCC 1 20 2 1 1 4
NciI CC/SGG 114 97 1 7 4 10
NcoI C/CATGG 4 20 0 0 0 0
NdeI CA/TATG 7 2 0 1 3 1
NdeII /GATC 116 87 0 15 7 22
NgoMIV G/CCGGC 1 13 0 0 1 4
NheI G/CTAGC 1 4 0 0 0 1
NotI GC/GGCCGC 0 7 0 0 0 0
NruI TCG/CGA 5 5 2 0 0 1
NsiI ATGCA/T 14 9 0 0 0 0
PstI CTGCA/G 28 30 1 1 1 1
PvuI CGAT/CG 3 7 0 2 1 1
PvuII CAG/CTG 15 24 0 2 3 1
RsaI GT/AC 113 83 11 3 19 3
SacI GAGCT/C 2 16 0 1 1 0
SacII CCGC/GG 4 33 1 0 0 0
Sal I G/TCGAC 2 3 0 1 1 1
Sau3AI G/ATC 116 87 0 15 7 22
Sau96I G/GNCC 74 164 2 6 4 15
ScaI AGT/ACT 5 5 0 1 0 1
SfiI GGCCNNNN/NGGCC 0 3 0 0 0 0
SgfI(a) GCGAT/CGC 0 1 0 0 0 0
SinI G/GWCC 35 73 1 2 1 8
SmaI CCC/GGG 3 12 0 1 1 0
SnaBI TAC/GTA 1 0 0 0 1 0
SpeI A/CTAGT 0 3 0 0 0 0
SphI GCATG/C 6 8 0 1 1 1
SspI AAT/ATT 20 5 1 1 6 1
StuI AGG/CCT 6 11 1 0 0 0
StyI C/CWWGG 10 44 0 0 0 1
TaqI T/CGA 121 50 10 4 13 7
Tru9I T/TAA 195 115 35 13 62 15
Tth111 I GACN/NNGTC 2 12 0 0 0 1
VspI AT/TAAT 17 3 2 3 6 1
XbaI T/CTAGA 1 5 0 1 1 0
XhoI C/TCGAG 1 6 1 0 0 0
XhoII R/GATCY 21 22 0 7 3 8
XmaI C/CCGGG 3 12 0 1 1 0
XmnI GAANN/NNTTC 24 5 3 1 2 2

(a)U.S. Pat. No. 5,391,487 has been issued to Promega Corporation for restriction endonuclease Sgf I.

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Multiple Cloning Sites of Promega Vectors

Promega's pGEM® Vectors and pSP Vectors are high copy number plasmid cloning vectors suitable for a wide variety of applications including routine cloning, sequencing, in vitro transcription and production of single-stranded DNA. Each vector contains a multiple cloning site featuring a variety of unique restriction enzyme sites. The multiple cloning sites of all these vectors except for the pSP64 Poly(A) Vector are flanked by SP6 and T7 phage RNA polymerase promoters. (The pSP64 Poly(A) Vector contains only the a SP6 phage promoter.) The opposing SP6 and T7 promoters allow in vitro transcription of either strand of an insert cloned into the multiple cloning site. In the pGEM® Vectors the multiple cloning site is embedded in a gene encoding the lacZ alpha-peptide, allowing selection of recombinants by blue/white color screening. The pGEM®-Zf Vectors also contain the origin of replication of the filamentous phage f1, a feature that allows synthesis of single-stranded DNA. See our vector page for more information on all of the plasmid vectors available from Promega.

Cloning Vector
pGEM®
3Z
pGEM®
4Z
pGEM®
3Zf
(+/-)
pGEM®
5Zf
(+/-)
pGEM®
7Zf
(+/-)
pGEM®
9Zf
(-)
pGEM®
11Zf
(+/-)
pGEM®
13Zf
(+)
pSP72 pSP73 pSP64
Poly(A)
AatII             x x                
AccI x x x                x x
Acc65I x x x    x          x   
ApaI          x x    x         
AvaI x x x                   x
BamHI x x x    x    x    x x
Bgl II                         x   
BstXI          x x               
BstZI          x    x x x      
ClaI             x          x   
Csp45I             x               
Eco       x   x x x    
EcoICRI x x x x x x x   x x
EcoRI x x x   x x x   x x
EcoRV       x         x  
HincII x x x             x
HindIII x x x   x x x x x x
KpnI x x x       x       x  
NcoI       x            
NdeI       x            
NotI       x   x x x    
NsiI       x x x x      
PstI x x x x         x x
PvuII                 x  
SacI x x x x x x x   x x
SacII       x            
SalI x x x x   x x   x x
SfiI           x x x    
SmaI x x x   x       x x
SpeI       x   x        
SphI x x x x x       x  
StyI       x            
Tth111 I           x        
XbaI x x x   x x x   x x
XhoI         x   x   x  

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Restriction Enzyme Recognition Sequences Containing Start and Stop Codons

Table 3.9A lists enzymes whose recognition sequences contain the start codon AUG. Those enzymes having a limited degree of degeneracy for the AUG codon (e.g., R, Y, W, etc.) are listed, however those that would require the AUG to be composed completely of unspecified nucleotides (e.g., NNN) are not included. These enzymes can be useful for creating promoter fusions in gene expression studies.

Table 3.9A. Enzymes that have the Start Codon (AUG) as Part of Their Recognition Sequence.
AflIII A/CRYGT BstF5I GGATG(2/0) NlaIII CATG/
BsaMI GAATGC(1/-1) DsaI C/CRYGG NsiI ATGCA/T
BseGI GGATG(2/0) FokI GGATG(9/13) NspI RCATG/Y
Bsp19I C/CATGG MslI CAYNN/NNRTG Ppu10I A/TGCAT
BspHI T/CATGA NcoI C/CATGG SphI GCATG/C
BspLU11 I A/CATGT NdeI CA/TATG StyI C/CWWGG
BsrDI GCAATG(2/0)        

Table 3.9B lists enzymes whose recognition sequences contain the stop codons UAA, UAG and UGA. Those enzymes having a limited degree of degeneracy for the stop codons (e.g. R, Y, W, etc.) are listed, however those that would require the stop codons to be composed completely of unspecified nucleotides (e.g., NNN) are not included.

Table 3.9B. Enzymes that Have a Stop Codon (UAA, UAG, UGA) as Part of Their Recognition Sequence.
UAA UAG UGA
AseI AT/TAAT AccI GT/MKAC AsuHPI GGTGA(8/7)
Bst98I C/TTAAG AvrII C/CTAGG Bcl I T/GATCA
BstHPI GTT/AAC BfaI C/TAG BspHI T/CATGA
DraI TTT/AAA BfmI C/TRYAG Eco57I CTGAAG(16/14)
HincII GTY/RAC NheI G/CTAGC HincII GTY/RAC
HpaI GTT/AAC SfcI C/TRYAG HphI GGTGA(8/7)
PacI TTAAT/TAA SpeI A/CTAGT SmlI C/TYRAG
PmeI GTTT/AAAC StyI C/CWWGG Tsp45I /GTSAG
SmlI C/TYRAC XbaI T/CTAGA    
SwaI ATTT/AAAT        
Tru9I T/TAA        
VspI AT/TAAT        

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IUPAC Ambiguity Codes for Nucleotide Degeneracy

R = A or G K = G or T S = G or C
Y = C or T M = A or C W = A or T
B = not A (C, G or T) H = not G (A, C or T) N = any nucleotide
D = not C (A, G or T) V = not T (A, C or G)  

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