pMAL-c5X

Bacterial vector for inducible cytoplasmic expression of maltose-binding protein (MBP) fusions with a Factor Xa cleavage site.

Sequence Author: New England Biolabs

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MscI * (5652) FspAI (5642) Bpu10I (5514) PflFI - Tth111I (4877) BspQI - SapI (4796) PciI (4679) Bsu36I (3990) BglI (3756) ScaI (3393) TatI (3391) DraI (3296) BspHI (3034) RsrII (2990) 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 (1818) PsiI (1880) BglII (1887) BmgBI (2070) BsmI (2148) BlpI (2314) Eco53kI (2636) SacI (2638) AvaI - BsoBI (2671) XmnI (2688) NdeI (2696) NcoI - StyI (2702) EagI - NotI (2709) EcoRV (2718) SalI - SgrDI (2722) AccI (2723) BamHI (2728) EcoRI (2734) PstI - SbfI (2745) HindIII (2757) pMAL-c5X 5677 bp
MscI  (5652)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
FspAI  (5642)
1 site
R T G C G C A Y Y A C G C G T R
Bpu10I  (5514)
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  (4877)
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  (4877)
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  (4796)
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  (4796)
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  (4679)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
Bsu36I  (3990)
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  (3756)
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  (3393)
1 site
A G T A C T T C A T G A
TatI  (3391)
1 site
W G T A C W W C A T G W
DraI  (3296)
1 site
T T T A A A A A A T T T
BspHI  (3034)
1 site
T C A T G A A G T A C T
RsrII  (2990)
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  (1818)
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  (1880)
1 site
T T A T A A A A T A T T
BglII  (1887)
1 site
A G A T C T T C T A G A
BmgBI  (2070)
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  (2148)
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  (2314)
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  (2636)
1 site
G A G C T C C T C G A G
SacI  (2638)
1 site
G A G C T C C T C G A G
AvaI  (2671)
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  (2671)
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.
XmnI  (2688)
1 site
G A A N N N N T T C C T T N N N N A A G
NdeI  (2696)
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  (2702)
1 site
C C A T G G G G T A C C
StyI  (2702)
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  (2709)
1 site
C G G C C G G C C G G C
NotI  (2709)
1 site
G C G G C C G C C G C C G G C G
EcoRV  (2718)
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  (2722)
1 site
G T C G A C C A G C T G
SgrDI  (2722)
1 site
C G T C G A C G G C A G C T G C
AccI  (2723)
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  (2728)
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  (2734)
1 site
G A A T T C C T T A A G
PstI  (2745)
1 site
C T G C A G G A C G T C
SbfI  (2745)
1 site
C C T G C A G G G G A C G T C C
HindIII  (2757)
1 site
A A G C T T T T C G A A
MBP
1528 .. 2628  =  1101 bp
367 amino acids  =  40.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
1528 .. 2628  =  1101 bp
367 amino acids  =  40.4 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
Factor Xa site
2677 .. 2688  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
Factor Xa site
2677 .. 2688  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa 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. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
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. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
3087 .. 3947  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   3087 .. 3155  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3087 .. 3947  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3156 .. 3947  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3087 .. 3947  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
4035 .. 4623  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4035 .. 4623  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
4993 .. 5184  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4993 .. 5184  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
2996 .. 3086  =  91 bp
AmpR promoter
2996 .. 3086  =  91 bp
rrnB T1 terminator
2763 .. 2849  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
2763 .. 2849  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
lacIq promoter
3 .. 80  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
3 .. 80  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
tac promoter
1406 .. 1434  =  29 bp
3 segments
   Segment 1:  -35  
   1406 .. 1411  =  6 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
tac promoter
1406 .. 1434  =  29 bp
3 segments
   Segment 2:  
   1412 .. 1427  =  16 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
tac promoter
1406 .. 1434  =  29 bp
3 segments
   Segment 3:  -10  
   1428 .. 1434  =  7 bp
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
tac promoter
1406 .. 1434  =  29 bp
3 segments
strong E. coli promoter; hybrid between the trp and lac UV5 promoters
rrnB T2 terminator
2941 .. 2968  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
2941 .. 2968  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
lac operator
1442 .. 1458  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1442 .. 1458  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
MCS
2684 .. 2762  =  79 bp
multiple cloning site
MCS
2684 .. 2762  =  79 bp
multiple cloning site
ORF:  1528 .. 2754  =  1227 bp
ORF:  408 amino acids  =  44.7 kDa
ORF:  14 .. 331  =  318 bp
ORF:  105 amino acids  =  11.2 kDa
ORF:  920 .. 1183  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5216 .. 5584  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  204 .. 1163  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3087 .. 3947  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  3551 .. 3817  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  946 .. 1197  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4993 .. 5217  =  225 bp
ORF:  74 a