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Plasmid Files

pCBG68-Basic

Promoterless vector for measuring the activity of promoter and enhancer sequences with a click beetle green luciferase assay.

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pCBG68-Basic.dna
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Sequence Author:  Promega
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BsgI (4801) AarI - BfuAI - BspMI (4773) RVprimer3 (4752 .. 4771) NotI (4643) DraIII (4297) XmnI (3744) AseI (3317) BpmI (3215) AhdI (3145) AlwNI (2668) PspFI (2560) BseYI (2556) Acc65I (1) KpnI (5) Eco53kI (9) SacI (11) MluI (15) NheI (21) BmtI (25) TspMI - XmaI (26) SmaI - SrfI (28) PaeR7I - XhoI (32) BglII (36) HindIII (53) NcoI - StyI (86) ZraI (216) AatII (218) BstBI (254) PasI (834) BclI * (932) MscI (1036) BlpI (1061) BmgBI (1476) AgeI (1657) XbaI (1734) FseI (1753) HpaI (1894) BsaBI * (1995) BamHI (1996) SalI (2002) PshAI (2067) RVprimer4 (2053 .. 2072) AfeI (2128) PciI (2252) NspI (2256) pCBG68-Basic 4810 bp
BsgI  (4801)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AarI  (4773)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
BfuAI  (4773)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (4773)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
NotI  (4643)
1 site
G C G G C C G C C G C C G G C G
DraIII  (4297)
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.
XmnI  (3744)
1 site
G A A N N N N T T C C T T N N N N A A G
AseI  (3317)
1 site
A T T A A T T A A T T A
BpmI  (3215)
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.
AhdI  (3145)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
AlwNI  (2668)
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  (2560)
1 site
C C C A G C G G G T C G
BseYI  (2556)
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.
Acc65I  (1)
1 site
G G T A C C C C A T G G
KpnI  (5)
1 site
G G T A C C C C A T G G
Eco53kI  (9)
1 site
G A G C T C C T C G A G
SacI  (11)
1 site
G A G C T C C T C G A G
MluI  (15)
1 site
A C G C G T T G C G C A
NheI  (21)
1 site
G C T A G C C G A T C G
BmtI  (25)
1 site
G C T A G C C G A T C G
TspMI  (26)
1 site
C C C G G G G G G C C C
XmaI  (26)
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  (28)
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  (28)
1 site
G C C C G G G C C G G G C C C G
PaeR7I  (32)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (32)
1 site
C T C G A G G A G C T C
BglII  (36)
1 site
A G A T C T T C T A G A
HindIII  (53)
1 site
A A G C T T T T C G A A
NcoI  (86)
1 site
C C A T G G G G T A C C
StyI  (86)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
ZraI  (216)
1 site
G A C G T C C T G C A G
AatII  (218)
1 site
G A C G T C C T G C A G
BstBI  (254)
1 site
T T C G A A A A G C T T
PasI  (834)
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.
BclI  (932)
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.
MscI  (1036)
1 site
T G G C C A A C C G G T
BlpI  (1061)
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.
BmgBI  (1476)
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  (1657)
1 site
A C C G G T T G G C C A
XbaI  (1734)
1 site
T C T A G A A G A T C T
FseI  (1753)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
HpaI  (1894)
1 site
G T T A A C C A A T T G
BsaBI  (1995)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
BamHI  (1996)
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.
SalI  (2002)
1 site
G T C G A C C A G C T G
PshAI  (2067)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
AfeI  (2128)
1 site
A G C G C T T C G C G A
PciI  (2252)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (2256)
1 site
R C A T G Y Y G T A C R
RVprimer3
20-mer  /  50% GC
1 binding site
4752 .. 4771  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
2053 .. 2072  =  20 annealed bases
Tm  =  61°C
CBG68luc
88 .. 1716  =  1629 bp
542 amino acids  =  60.2 kDa
Product: green-emitting variant of click beetle luciferase
human codon-optimized
CBG68luc
88 .. 1716  =  1629 bp
542 amino acids  =  60.2 kDa
Product: green-emitting variant of click beetle luciferase
human codon-optimized
AmpR
3072 .. 3932  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3072 .. 3863  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3072 .. 3932  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3864 .. 3932  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3072 .. 3932  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2313 .. 2901  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2313 .. 2901  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4064 .. 4519  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4064 .. 4519  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 poly(A) signal
1773 .. 1894  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1773 .. 1894  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3933 .. 4037  =  105 bp
AmpR promoter
3933 .. 4037  =  105 bp
pause site
4712 .. 4803  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
pause site
4712 .. 4803  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
MCS
1 .. 58  =  58 bp
multiple cloning site
MCS
1 .. 58  =  58 bp
multiple cloning site
poly(A) signal
4650 .. 4698  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4650 .. 4698  =  49 bp
synthetic polyadenylation signal
ORF:  88 .. 1716  =  1629 bp
ORF:  542 amino acids  =  60.2 kDa
ORF:  3202 .. 3468  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1859 .. 2101  =  243 bp
ORF:  80 amino acids  =  9.0 kDa
ORF:  22 .. 633  =  612 bp
ORF:  203 amino acids  =  23.1 kDa
ORF:  946 .. 1728  =  783 bp
ORF:  260 amino acids  =  29.0 kDa
ORF:  4597 .. 89  =  303 bp
ORF:  100 amino acids  =  11.9 kDa
ORF:  3072 .. 3932  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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