pBAC-3

Baculovirus transfer plasmid with the polyhedrin promoter, encoding an N-terminal signal sequence-6xHis-thrombin-S-Tag-enterokinase cassette.

Sequence Author: MilliporeSigma (Novagen)

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DraIII (5182) ScaI (4510) NmeAIII (4178) BpmI (4100) BmrI (4070) AlwNI (3553) PspFI (3445) BseYI (3441) PciI (3137) PmeI (3017) SgrAI (2729) BstXI (315) NsiI (332) MluI (547) BbvCI - Bpu10I (564) baculovirus recombination region (lef2/ORF603) BglII (1161) BsaBI (1192) NcoI (1327) 6xHis SacII (1357) thrombin site PflMI (1418) NheI (1428) BmtI (1432) enterokinase site BseRI - TspMI - XmaI (1460) SmaI - SrfI (1462) StuI * (1480) BamHI (1484) EcoRI (1490) Eco53kI (1498) SacI (1500) HindIII (1503) EagI - NotI (1510) PaeR7I - PspXI - XhoI (1518) 6xHis AvrII (1545) BlpI (1554) SphI (1564) SnaBI (1641) SwaI (1731) ZraI (1920) AatII (1922) BmgBI (2145) AgeI (2489) AleI (2720) pBAC™-3 5474 bp
DraIII  (5182)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
ScaI  (4510)
1 site
A G T A C T T C A T G A
NmeAIII  (4178)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BpmI  (4100)
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.
BmrI  (4070)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
AlwNI  (3553)
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  (3445)
1 site
C C C A G C G G G T C G
BseYI  (3441)
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.
PciI  (3137)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PmeI  (3017)
1 site
G T T T A A A C C A A A T T T G
SgrAI  (2729)
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.
BstXI  (315)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
NsiI  (332)
1 site
A T G C A T T A C G T A
MluI  (547)
1 site
A C G C G T T G C G C A
BbvCI  (564)
1 site
C C T C A G C G G A G T C G
Bpu10I  (564)
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.
BglII  (1161)
1 site
A G A T C T T C T A G A
BsaBI  (1192)
1 site
G A T N N N N A T C C T A N N N N T A G
NcoI  (1327)
1 site
C C A T G G G G T A C C
SacII  (1357)
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.
PflMI  (1418)
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.
NheI  (1428)
1 site
G C T A G C C G A T C G
BmtI  (1432)
1 site
G C T A G C C G A T C G
BseRI  (1460)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
TspMI  (1460)
1 site
C C C G G G G G G C C C
XmaI  (1460)
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  (1462)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (1462)
1 site
G C C C G G G C C G G G C C C G
StuI  (1480)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BamHI  (1484)
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  (1490)
1 site
G A A T T C C T T A A G
Eco53kI  (1498)
1 site
G A G C T C C T C G A G
SacI  (1500)
1 site
G A G C T C C T C G A G
HindIII  (1503)
1 site
A A G C T T T T C G A A
EagI  (1510)
1 site
C G G C C G G C C G G C
NotI  (1510)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (1518)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1518)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1518)
1 site
C T C G A G G A G C T C
AvrII  (1545)
1 site
C C T A G G G G A T C C
BlpI  (1554)
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.
SphI  (1564)
1 site
G C A T G C C G T A C G
SnaBI  (1641)
1 site
T A C G T A A T G C A T
SwaI  (1731)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
ZraI  (1920)
1 site
G A C G T C C T G C A G
AatII  (1922)
1 site
G A C G T C C T G C A G
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.
AgeI  (2489)
1 site
A C C G G T T G G C C A
AleI  (2720)
1 site
C A C N N N N G T G G T G N N N N C A C
baculovirus recombination region (ORF1629)
1578 .. 2935  =  1358 bp
contains part of ORF1629
baculovirus recombination region (ORF1629)
1578 .. 2935  =  1358 bp
contains part of ORF1629
AmpR
3957 .. 4817  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3957 .. 4748  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3957 .. 4817  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4749 .. 4817  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3957 .. 4817  =  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)
329 .. 1156  =  828 bp
contains ORF603 and part of lef2
baculovirus recombination region (lef2/ORF603)
329 .. 1156  =  828 bp
contains ORF603 and part of lef2
ori
3198 .. 3786  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3198 .. 3786  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4949 .. 5404  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4949 .. 5404  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
GP64 signal sequence
1266 .. 1325  =  60 bp
20 amino acids  =  2.0 kDa
Product: signal sequence from the baculovirus envelope glycoprotein GP64
GP64 signal sequence
1266 .. 1325  =  60 bp
20 amino acids  =  2.0 kDa
Product: signal sequence from the baculovirus envelope glycoprotein GP64
6xHis
1335 .. 1352  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1335 .. 1352  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
thrombin site
1362 .. 1379  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
1362 .. 1379  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
S-Tag
1383 .. 1427  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
1383 .. 1427  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
enterokinase site
1443 .. 1457  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
1443 .. 1457  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
6xHis
1524 .. 1541  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1524 .. 1541  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
AmpR promoter
4818 .. 4922  =  105 bp
AmpR promoter
4818 .. 4922  =  105 bp
polyhedrin promoter
1168 .. 1259  =  92 bp
promoter for the baculovirus polyhedrin gene
polyhedrin promoter
1168 .. 1259  =  92 bp
promoter for the baculovirus polyhedrin gene
MCS
1460 .. 1523  =  64 bp
multiple cloning site
MCS
1460 .. 1523  =  64 bp
multiple cloning site
ORF1629
1594 .. 2935  =  1342 bp
446 amino acids  =  49.2 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF1629
1594 .. 2935  =  1342 bp
446 amino acids  =  49.2 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF603
491 .. 1096  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF603
491 .. 1096  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
lef2
329 .. 453  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
lef2
329 .. 453  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
ORF:  73 .. 453  =  381 bp
ORF:  126 amino acids  =  14.4 kDa
ORF:  1288 .. 1557  =  270 bp
ORF:  89 amino acids  =  10.4 kDa
ORF:  4087 .. 4353  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1260 .. 1544  =  285 bp
ORF:  94 amino acids  =  9.9 kDa
ORF:  3957 .. 4817  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  491 .. 1096  =  606 bp
ORF:  201 amino acids  =  23.6 kDa
ORF:  535 .. 765  =  231 bp
ORF:  76 amino acids  =  9.2 kDa
ORF:  1594 .. 2847  =  1254 bp
ORF:  417 amino acids  =  46.0 kDa
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