pAB-MBP

Baculovirus transfer plasmid for high-level expression of proteins with a thrombin-cleavable N-terminal MBP tag.

Sequence Author: AB Vector

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NdeI (10,579) ScaI (9888) BpmI (9478) BspQI - SapI (8399) FspAI (7482) SgrDI (7329) SgrAI (7060) baculovirus recombination region (ORF1629) SnaBI (5972) KpnI (5792) Acc65I (5788) PasI (486) PacI (583) XcmI (746) SacII (871) BstEII (923) PspOMI (1395) ApaI (1399) StyI (1501) PaeR7I - PspXI - XhoI (1901) SphI (2135) BbvCI - Bpu10I (3405) baculovirus recombination region (lef2/ORF603) NgoMIV (3770) NaeI (3772) EcoRV (4000) BamHI (4129) BssHII (4579) BlpI (4929) Eco53kI (5251) SacI (5253) StuI (5260) XbaI (5279) EcoRI (5290) TspMI - XmaI (5302) SmaI (5304) EagI - NotI (5313) KflI - PpuMI (5341) pAB-MBP™ 10,793 bp
NdeI  (10,579)
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.
ScaI  (9888)
1 site
A G T A C T T C A T G A
BpmI  (9478)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BspQI  (8399)
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  (8399)
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.
FspAI  (7482)
1 site
R T G C G C A Y Y A C G C G T R
SgrDI  (7329)
1 site
C G T C G A C G G C A G C T G C
SgrAI  (7060)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
SnaBI  (5972)
1 site
T A C G T A A T G C A T
KpnI  (5792)
1 site
G G T A C C C C A T G G
Acc65I  (5788)
1 site
G G T A C C C C A T G G
PasI  (486)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PacI  (583)
1 site
T T A A T T A A A A T T A A T T
XcmI  (746)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
SacII  (871)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
BstEII  (923)
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  (1395)
1 site
G G G C C C C C C G G G
ApaI  (1399)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
StyI  (1501)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PaeR7I  (1901)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1901)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1901)
1 site
C T C G A G G A G C T C
SphI  (2135)
1 site
G C A T G C C G T A C G
BbvCI  (3405)
1 site
C C T C A G C G G A G T C G
Bpu10I  (3405)
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.
NgoMIV  (3770)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (3772)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
EcoRV  (4000)
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.
BamHI  (4129)
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.
BssHII  (4579)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BlpI  (4929)
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  (5251)
1 site
G A G C T C C T C G A G
SacI  (5253)
1 site
G A G C T C C T C G A G
StuI  (5260)
1 site
A G G C C T T C C G G A
XbaI  (5279)
1 site
T C T A G A A G A T C T
EcoRI  (5290)
1 site
G A A T T C C T T A A G
TspMI  (5302)
1 site
C C C G G G G G G C C C
XmaI  (5302)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (5304)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
EagI  (5313)
1 site
C G G C C G G C C G G C
NotI  (5313)
1 site
G C G G C C G C C G C C G G C G
KflI  (5341)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (5341)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
MBP
4146 .. 5243  =  1098 bp
366 amino acids  =  40.2 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
4146 .. 5243  =  1098 bp
366 amino acids  =  40.2 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.
thrombin site
5262 .. 5279  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
5262 .. 5279  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
AmpR
9335 .. 10,195  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   9335 .. 10,126  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
9335 .. 10,195  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   10,127 .. 10,195  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
9335 .. 10,195  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
baculovirus recombination region (lef2/ORF603)
3170 .. 3997  =  828 bp
contains ORF603 and part of lef2
baculovirus recombination region (lef2/ORF603)
3170 .. 3997  =  828 bp
contains ORF603 and part of lef2
baculovirus recombination region (ORF1629)
5808 .. 6513  =  706 bp
contains part of ORF1629
baculovirus recombination region (ORF1629)
5808 .. 6513  =  706 bp
contains part of ORF1629
ori
8576 .. 9164  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
8576 .. 9164  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
10,196 .. 10,300  =  105 bp
AmpR promoter
10,196 .. 10,300  =  105 bp
polyhedrin promoter
4001 .. 4092  =  92 bp
promoter for the baculovirus polyhedrin gene
polyhedrin promoter
4001 .. 4092  =  92 bp
promoter for the baculovirus polyhedrin gene
ORF603
3332 .. 3937  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF603
3332 .. 3937  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF1629
5925 .. 6513  =  589 bp
195 amino acids  =  22.3 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF1629
5925 .. 6513  =  589 bp
195 amino acids  =  22.3 kDa
Product: baculovirus capsid-associated protein
required for viral replication
lef2
3170 .. 3294  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
lef2
3170 .. 3294  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
MCS
5249 .. 5346  =  98 bp
multiple cloning site
MCS
5249 .. 5346  =  98 bp
multiple cloning site
ORF:  76 .. 582  =  507 bp
ORF:  168 amino acids  =  19.3 kDa
ORF:  2662 .. 3294  =  633 bp
ORF:  210 amino acids  =  23.9 kDa
ORF:  5284 .. 5895  =  612 bp
ORF:  203 amino acids  =  23.5 kDa
ORF:  7549 .. 8139  =  591 bp
ORF:  196 amino acids  =  22.9 kDa
ORF:  7973 .. 8251  =  279 bp
ORF:  92 amino acids  =  10.5 kDa
ORF:  969 .. 1241  =  273 bp
ORF:  90 amino acids  =  9.7 kDa
ORF:  2352 .. 2681  =  330 bp
ORF:  109 amino acids  =  12.4 kDa
ORF:  4146 .. 5330  =  1185 bp
ORF:  394 amino acids  =  43.0 kDa
ORF:  9465 .. 9731  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  10,581 .. 15  =  228 bp
ORF:  75 amino acids  =  8.8 kDa
ORF:  5925 .. 7550  =  1626 bp
ORF:  541 amino acids  =  60.5 kDa
ORF:  614 .. 1600  =  987 bp
ORF:  328 amino acids  =  37.8 kDa
ORF:  1868 .. 2323  =  456 bp
ORF:  151 amino acids  =  17.6 kDa
ORF:  3332 .. 3937  =  606 bp
ORF:  201 amino acids  =  23.6 kDa
ORF:  9335 .. 10,195  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3376 .. 3606  =  231 bp
ORF:  76 amino acids  =  9.3 kDa
ORF:  5242 .. 5607  =  366 bp
ORF:  121 amino acids  =  13.3 kDa
ORF:  5632 .. 5907  =  276 bp
ORF:  91 amino acids  =  10.5 kDa
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