pGL3-Basic

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

Sequence Author: Promega

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Eco53kI (9) KpnI (5) Acc65I (1) BsgI (4809) RVprimer3 (4760 .. 4779) poly(A) signal NotI (4651) DraIII (4305) BsaAI (4302) BtgZI (4297) XmnI (3752) AseI (3325) NmeAIII (3301) BsaI (3214) AhdI (3153) AlwNI (2676) SacI (11) MluI (15) NheI (21) BmtI (25) TspMI - XmaI (26) SmaI - SrfI (28) MCS PaeR7I - XhoI (32) BglII (36) HindIII (53) NcoI - StyI (86) GLprimer2 (89 .. 111) KasI (120) NarI (121) SfoI (122) PluTI (124) PfoI * (195) BstBI (257) BsrGI (578) BclI * (668) SphI (751) XcmI (823) EcoO109I - PpuMI (1267) SgrAI (1516) XbaI (1742) FseI (1761) HpaI (1902) BsaBI * (2003) BamHI (2004) SalI (2010) AccI (2011) PshAI (2075) RVprimer4 (2061 .. 2080) AfeI (2136) PciI (2260) pGL3-Basic 4818 bp
Eco53kI  (9)
1 site
G A G C T C C T C G A G
KpnI  (5)
1 site
G G T A C C C C A T G G
Acc65I  (1)
1 site
G G T A C C C C A T G G
BsgI  (4809)
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).
NotI  (4651)
1 site
G C G G C C G C C G C C G G C G
DraIII  (4305)
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.
BsaAI  (4302)
1 site
Y A C G T R R T G C A Y
BtgZI  (4297)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
XmnI  (3752)
1 site
G A A N N N N T T C C T T N N N N A A G
AseI  (3325)
1 site
A T T A A T T A A T T A
NmeAIII  (3301)
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).
BsaI  (3214)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (3153)
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  (2676)
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.
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

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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.
KasI  (120)
1 site
G G C G C C C C G C G G
NarI  (121)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (122)
1 site
G G C G C C C C G C G G
PluTI  (124)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
PfoI  (195)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BstBI  (257)
1 site
T T C G A A A A G C T T
BsrGI  (578)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BclI  (668)
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.
SphI  (751)
1 site
G C A T G C C G T A C G
XcmI  (823)
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.
EcoO109I  (1267)
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.
PpuMI  (1267)
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.
SgrAI  (1516)
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.
XbaI  (1742)
1 site
T C T A G A A G A T C T
FseI  (1761)
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  (1902)
1 site
G T T A A C C A A T T G
BsaBI  (2003)
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  (2004)
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  (2010)
1 site
G T C G A C C A G C T G
AccI  (2011)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
PshAI  (2075)
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  (2136)
1 site
A G C G C T T C G C G A
PciI  (2260)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
RVprimer3
20-mer  /  50% GC
1 binding site
4760 .. 4779  =  20 annealed bases
Tm  =  54°C
GLprimer2
23-mer  /  39% GC
1 binding site
89 .. 111  =  23 annealed bases
Tm  =  57°C
RVprimer4
20-mer  /  65% GC
1 binding site
2061 .. 2080  =  20 annealed bases
Tm  =  62°C
luciferase
88 .. 1740  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
enhanced luc+ version of the luciferase gene
luciferase
88 .. 1740  =  1653 bp
550 amino acids  =  60.6 kDa
Product: firefly luciferase
enhanced luc+ version of the luciferase gene
AmpR
3080 .. 3940  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3080 .. 3871  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3080 .. 3940  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3872 .. 3940  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3080 .. 3940  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2321 .. 2909  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2321 .. 2909  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
f1 ori
4072 .. 4527  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4072 .. 4527  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 poly(A) signal
1781 .. 1902  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1781 .. 1902  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3941 .. 4045  =  105 bp
AmpR promoter
3941 .. 4045  =  105 bp
pause site
4720 .. 4811  =  92 bp
RNA polymerase II transcriptional pause signal from the human α2 globin gene
pause site
4720 .. 4811  =  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
4658 .. 4706  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4658 .. 4706  =  49 bp
synthetic polyadenylation signal
ORF:  88 .. 1740  =  1653 bp
ORF:  550 amino acids  =  60.6 kDa
ORF:  3210 .. 3476  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  373 .. 600  =  228 bp
ORF:  75 amino acids  =  9.1 kDa
ORF:  1867 .. 2109  =  243 bp
ORF:  80 amino acids  =  9.0 kDa
ORF:  4605 .. 107  =  321 bp
ORF:  106 amino acids  =  12.5 kDa
ORF:  3080 .. 3940  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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