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Plasmid Files

pAcHLT C

Baculovirus transfer vector for expressing proteins with an N-terminal 6xHis-PKA-thrombin cassette. For other reading frames, use pAcHLT A or pAcHLT B.

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pAcHLT C Sequence and MappAcHLT C.dna
Map and Sequence File   
Sequence Author:  BD Biosciences
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 BspQI - SapI (7865) PspFI (7448) BseYI (7444) AlwNI (7339) BpmI (6791) NmeAIII (6713) ScaI (6379) EcoO109I (5880) PfoI (5821) DraIII (5464) MscI (4154) FspAI (4084) SphI (234) BclI * (331) BstXI (1255) BbvCI - Bpu10I (1504) baculovirus recombination region (lef2/ORF603) EcoRV (2099) PKA site thrombin site NdeI (2314) PaeR7I - PspXI - XhoI (2319) EcoRI (2325) StuI (2333) BtgI - NcoI - StyI (2337) Eco53kI (2345) SacI (2347) NotI (2350) PstI - SbfI (2361) Acc65I (2363) KpnI (2367) TspMI - XmaI (2369) SmaI (2371) BglII (2375) SnaBI (2564) SwaI (2654) AgeI (3412) SgrAI (3652) SgrDI (3931) pAcHLT C 8108 bp
BspQI  (7865)
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  (7865)
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.
PspFI  (7448)
1 site
C C C A G C G G G T C G
BseYI  (7444)
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.
AlwNI  (7339)
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.
BpmI  (6791)
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.
NmeAIII  (6713)
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).
ScaI  (6379)
1 site
A G T A C T T C A T G A
EcoO109I  (5880)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
PfoI  (5821)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
DraIII  (5464)
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.
MscI  (4154)
1 site
T G G C C A A C C G G T
FspAI  (4084)
1 site
R T G C G C A Y Y A C G C G T R
SphI  (234)
1 site
G C A T G C C G T A C G
BclI  (331)
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.
BstXI  (1255)
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.
BbvCI  (1504)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1504)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
EcoRV  (2099)
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.
NdeI  (2314)
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.
PaeR7I  (2319)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2319)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2319)
1 site
C T C G A G G A G C T C
EcoRI  (2325)
1 site
G A A T T C C T T A A G
StuI  (2333)
1 site
A G G C C T T C C G G A
BtgI  (2337)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (2337)
1 site
C C A T G G G G T A C C
StyI  (2337)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
Eco53kI  (2345)
1 site
G A G C T C C T C G A G
SacI  (2347)
1 site
G A G C T C C T C G A G
NotI  (2350)
1 site
G C G G C C G C C G C C G G C G
PstI  (2361)
1 site
C T G C A G G A C G T C
SbfI  (2361)
1 site
C C T G C A G G G G A C G T C C
Acc65I  (2363)
1 site
G G T A C C C C A T G G
KpnI  (2367)
1 site
G G T A C C C C A T G G
TspMI  (2369)
1 site
C C C G G G G G G C C C
XmaI  (2369)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (2371)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BglII  (2375)
1 site
A G A T C T T C T A G A
SnaBI  (2564)
1 site
T A C G T A A T G C A T
SwaI  (2654)
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.
AgeI  (3412)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
SgrAI  (3652)
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.
SgrDI  (3931)
1 site
C G T C G A C G G C A G C T G C
baculovirus recombination region (ORF1629)
2400 .. 3858  =  1459 bp
contains part of ORF1629
baculovirus recombination region (ORF1629)
2400 .. 3858  =  1459 bp
contains part of ORF1629
AmpR
6073 .. 6933  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6073 .. 6141  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6073 .. 6933  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6142 .. 6933  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6073 .. 6933  =  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)
1269 .. 2096  =  828 bp
contains ORF603 and part of lef2
baculovirus recombination region (lef2/ORF603)
1269 .. 2096  =  828 bp
contains ORF603 and part of lef2
ori
7104 .. 7692  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7104 .. 7692  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5231 .. 5686  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5231 .. 5686  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
AmpR promoter
5968 .. 6072  =  105 bp
AmpR promoter
5968 .. 6072  =  105 bp
polyhedrin promoter
2100 .. 2191  =  92 bp
promoter for the baculovirus polyhedrin gene
polyhedrin promoter
2100 .. 2191  =  92 bp
promoter for the baculovirus polyhedrin gene
MCS
2313 .. 2380  =  68 bp
multiple cloning site
MCS
2313 .. 2380  =  68 bp
multiple cloning site
6xHis
2230 .. 2247  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2230 .. 2247  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
PKA site
2254 .. 2271  =  18 bp
6 amino acids  =  658.8 Da
Product: recognition site for phosphorylation by
protein kinase A
PKA site
2254 .. 2271  =  18 bp
6 amino acids  =  658.8 Da
Product: recognition site for phosphorylation by
protein kinase A
thrombin site
2281 .. 2298  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
2281 .. 2298  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
ORF1629
2517 .. 3858  =  1342 bp
446 amino acids  =  49.2 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF1629
2517 .. 3858  =  1342 bp
446 amino acids  =  49.2 kDa
Product: baculovirus capsid-associated protein
required for viral replication
ORF603
1431 .. 2036  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
ORF603
1431 .. 2036  =  606 bp
201 amino acids  =  23.6 kDa
Product: baculovirus ORF603 protein
lef2
1269 .. 1393  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
lef2
1269 .. 1393  =  125 bp
40 amino acids  =  4.5 kDa
Product: baculovirus late expression factor 2
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