pMAL-p5E

Bacterial vector for inducible periplasmic expression of maltose-binding protein (MBP) fusions with an enterokinase cleavage site.

Sequence Author: New England Biolabs

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MscI * (5730) FspAI (5720) Bpu10I (5592) PflFI - Tth111I (4955) BspQI - SapI (4874) PciI (4757) Bsu36I (4068) BglI (3834) ScaI (3471) TatI (3469) DraI (3374) BspHI (3112) RsrII (3068) PflMI (13) MluI (431) BstEII (612) PspOMI (638) ApaI (642) HpaI (937) KasI (1070) NarI * (1071) SfoI (1072) PluTI (1074) lac operator MfeI (1502) BsiWI (1893) PsiI (1955) BglII (1962) BmgBI (2145) BsmI (2223) BlpI (2389) Eco53kI (2711) SacI (2713) AvaI - BsoBI (2746) enterokinase site Acc65I (2766) KpnI (2770) NdeI (2774) NcoI - StyI (2780) EagI - NotI (2787) EcoRV (2796) SalI - SgrDI (2800) AccI (2801) BamHI (2806) EcoRI (2812) PstI - SbfI (2823) HindIII (2835) pMAL-p5E 5755 bp
MscI  (5730)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
FspAI  (5720)
1 site
R T G C G C A Y Y A C G C G T R
Bpu10I  (5592)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PflFI  (4955)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (4955)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BspQI  (4874)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (4874)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
PciI  (4757)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
Bsu36I  (4068)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BglI  (3834)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
ScaI  (3471)
1 site
A G T A C T T C A T G A
TatI  (3469)
1 site
W G T A C W W C A T G W
DraI  (3374)
1 site
T T T A A A A A A T T T
BspHI  (3112)
1 site
T C A T G A A G T A C T
RsrII  (3068)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
PflMI  (13)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
MluI  (431)
1 site
A C G C G T T G C G C A
BstEII  (612)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
PspOMI  (638)
1 site
G G G C C C C C C G G G
ApaI  (642)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
HpaI  (937)
1 site
G T T A A C C A A T T G
KasI  (1070)
1 site
G G C G C C C C G C G G
NarI  (1071)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (1072)
1 site
G G C G C C C C G C G G
PluTI  (1074)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
MfeI  (1502)
1 site
C A A T T G G T T A A C
BsiWI  (1893)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PsiI  (1955)
1 site
T T A T A A A A T A T T
BglII  (1962)
1 site
A G A T C T T C T A G A
BmgBI  (2145)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
BsmI  (2223)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BlpI  (2389)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Eco53kI  (2711)
1 site
G A G C T C C T C G A G
SacI  (2713)
1 site
G A G C T C C T C G A G
AvaI  (2746)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2746)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
Acc65I  (2766)
1 site
G G T A C C C C A T G G
KpnI  (2770)
1 site
G G T A C C C C A T G G
NdeI  (2774)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
NcoI  (2780)
1 site
C C A T G G G G T A C C
StyI  (2780)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
EagI  (2787)
1 site
C G G C C G G C C G G C
NotI  (2787)
1 site
G C G G C C G C C G C C G G C G
EcoRV  (2796)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
SalI  (2800)
1 site
G T C G A C C A G C T G
SgrDI  (2800)
1 site
C G T C G A C G G C A G C T G C
AccI  (2801)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BamHI  (2806)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
EcoRI  (2812)
1 site
G A A T T C C T T A A G
PstI  (2823)
1 site
C T G C A G G A C G T C
SbfI  (2823)
1 site
C C T G C A G G G G A C G T C C
HindIII  (2835)
1 site
A A G C T T T T C G A A
MBP
1528 .. 2703  =  1176 bp
392 amino acids  =  42.9 kDa
2 segments
   Segment 1:  signal sequence  
   1528 .. 1605  =  78 bp
   26 amino acids  =  2.7 kDa
Product: maltose binding protein from E. coli
This gene encodes a signal sequence that can direct MBP to the bacterial periplasm.
MBP
1528 .. 2703  =  1176 bp
392 amino acids  =  42.9 kDa
2 segments
   Segment 2:  
   1606 .. 2703  =  1098 bp
   366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This gene encodes a signal sequence that can direct MBP to the bacterial periplasm.
MBP
1528 .. 2703  =  1176 bp
392 amino acids  =  42.9 kDa
2 segments
Product: maltose binding protein from E. coli
This gene encodes a signal sequence that can direct MBP to the bacterial periplasm.
enterokinase site
2752 .. 2766  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
2752 .. 2766  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
lacI
81 .. 1163  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli