pAB-bee-8xHis

Baculovirus transfer plasmid for high-level expression of secreted proteins with N- and C-terminal 8xHis tags.

Sequence Author: AB Vector

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NdeI (9751) ScaI (9060) AlwNI (8103) PspFI (7995) BseYI (7991) BspQI - SapI (7571) FspAI (6654) SgrDI (6501) SgrAI (6232) baculovirus recombination region (ORF1629) SnaBI (5144) PacI (583) XcmI (746) BtgI (868) SacII (871) BstEII (923) PspOMI (1395) ApaI - BanII (1399) PaeR7I - PspXI - XhoI (1901) SphI (2135) BclI * (2232) BbvCI - Bpu10I (3405) baculovirus recombination region (lef2/ORF603) NgoMIV (3770) NaeI (3772) EcoRV (4000) BglII (4134) melittin signal sequence BstZ17I (4184) 8xHis EagI - NotI (4237) XbaI (4252) TspMI - XmaI (4263) SmaI (4265) BamHI (4267) AvrII (4488) KflI - PpuMI (4513) pAB-bee™-8xHis 9965 bp
NdeI  (9751)
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  (9060)
1 site
A G T A C T T C A T G A
AlwNI  (8103)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PspFI  (7995)
1 site
C C C A G C G G G T C G
BseYI  (7991)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
BspQI  (7571)
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  (7571)
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  (6654)
1 site
R T G C G C A Y Y A C G C G T R
SgrDI  (6501)
1 site
C G T C G A C G G C A G C T G C
SgrAI  (6232)
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  (5144)
1 site
T A C G T A A T G C A T
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.
BtgI  (868)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI 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.
BanII  (1399)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII 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
BclI  (2232)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
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.
BglII  (4134)
1 site
A G A T C T T C T A G A
BstZ17I  (4184)
1 site
G T A T A C C A T A T G
EagI  (4237)
1 site
C G G C C G G C C G G C
NotI  (4237)
1 site
G C G G C C G C C G C C G G C G
XbaI  (4252)
1 site
T C T A G A A G A T C T
TspMI  (4263)
1 site
C C C G G G G G G C C C
XmaI  (4263)
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  (4265)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (4267)
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.
AvrII  (4488)
1 site
C C T A G G G G A T C C
KflI  (4513)
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  (4513)
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.
AmpR
8507 .. 9367  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   8507 .. 9298  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8507 .. 9367  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   9299 .. 9367  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
8507 .. 9367  =  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)
4980 .. 5685  =  706 bp
contains part of ORF1629
baculovirus recombination region (ORF1629)
4980 .. 5685  =  706 bp
contains part of ORF1629
ori
7748 .. 8336  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7748 .. 8336  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
MCS
4236 .. 4518  =  283 bp
multiple cloning site
MCS
4236 .. 4518  =  283 bp
multiple cloning site
AmpR promoter
9368 .. 9472  =  105 bp
AmpR promoter
9368 .. 9472  =  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
melittin signal sequence
4143 .. 4205  =  63 bp
21 amino acids  =  2.5 kDa
Product: signal sequence from honeybee melittin
melittin signal sequence
4143 .. 4205  =  63 bp
21 amino acids  =  2.5 kDa
Product: signal sequence from honeybee melittin
8xHis
4209 .. 4232  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
4209 .. 4232  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
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:  4576 .. 5067  =  492 bp
ORF:  163 amino acids  =  19.1 kDa
ORF:  6721 .. 7311  =  591 bp
ORF:  196 amino acids  =  22.9 kDa
ORF:  7145 .. 7423  =  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:  8637 .. 8903  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  9753 .. 15  =  228 bp
ORF:  75 amino acids  =  8.8 kDa
ORF:  5097 .. 6722  =  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:  8507 .. 9367  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3376 .. 3606  =  231 bp
ORF:  76 amino acids  =  9.3 kDa
ORF:  4489 .. 4779  =  291 bp
ORF:  96 amino acids  =  10.6 kDa
ORF:  4804 .. 5079  =  276 bp
ORF:  91 amino acids  =  10.5 kDa
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