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Plasmid Files

phRG-TK

Mammalian vector for weak constitutive expression of humanized Renilla luciferase.

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phRG-TK Sequence and MapphRG-TK.dna
Map and Sequence File   
Sequence Author:  Promega
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 BsgI (4834) AarI - BfuAI - BspMI (4806) RVprimer3 (4785 .. 4804) NotI (4676) BtgZI (4322) AseI (3350) AhdI (3178) Acc65I (1) KpnI (5) Eco53kI (9) SacI (11) NheI (21) BmtI (25) SrfI (28) PaeR7I - XhoI (32) BglII (36) MscI (163) BssHII (276) KasI (324) NarI (325) SfoI (326) PluTI (328) SacII (334) PpuMI (354) AvrII (357) BstZ17I (378) PvuII (566) EcoRI (684) BstBI (688) HindIII (795) BstAPI (1018) NsiI (1049) PfoI * (1110) EcoRV (1315) Bsu36I (1416) NruI (1491) ZraI (1525) PflFI - Tth111I (1526) AatII (1527) PasI (1604) XbaI (1767) FseI (1786) HpaI (1927) MfeI (1936) BamHI (2029) SalI (2035) PshAI (2100) RVprimer4 (2086 .. 2105) AfeI (2161) PciI (2285) phRG-TK 4843 bp
BsgI  (4834)
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  (4806)
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  (4806)
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  (4806)
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  (4676)
1 site
G C G G C C G C C G C C G G C G
BtgZI  (4322)
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.
AseI  (3350)
1 site
A T T A A T T A A T T A
AhdI  (3178)
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.
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
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
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
MscI  (163)
1 site
T G G C C A A C C G G T
BssHII  (276)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
KasI  (324)
1 site
G G C G C C C C G C G G
NarI  (325)
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  (326)
1 site
G G C G C C C C G C G G
PluTI  (328)
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.
SacII  (334)
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.
PpuMI  (354)
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.
AvrII  (357)
1 site
C C T A G G G G A T C C
BstZ17I  (378)
1 site
G T A T A C C A T A T G
PvuII  (566)
1 site
C A G C T G G T C G A C
EcoRI  (684)
1 site
G A A T T C C T T A A G
BstBI  (688)
1 site
T T C G A A A A G C T T
HindIII  (795)
1 site
A A G C T T T T C G A A
BstAPI  (1018)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
NsiI  (1049)
1 site
A T G C A T T A C G T A
PfoI  (1110)
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.
EcoRV  (1315)
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.
Bsu36I  (1416)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
NruI  (1491)
1 site
T C G C G A A G C G C T
ZraI  (1525)
1 site
G A C G T C C T G C A G
PflFI  (1526)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (1526)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
AatII  (1527)
1 site
G A C G T C C T G C A G
PasI  (1604)
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.
XbaI  (1767)
1 site
T C T A G A A G A T C T
FseI  (1786)
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  (1927)
1 site
G T T A A C C A A T T G
MfeI  (1936)
1 site
C A A T T G G T T A A C
BamHI  (2029)
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  (2035)
1 site
G T C G A C C A G C T G
PshAI  (2100)
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  (2161)
1 site
A G C G C T T C G C G A
PciI  (2285)
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
4785 .. 4804  =  20 annealed bases
Tm  =  54°C
RVprimer4
20-mer  /  65% GC
1 binding site
2086 .. 2105  =  20 annealed bases
Tm  =  61°C
hRluc
830 .. 1765  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
hRluc
830 .. 1765  =  936 bp
311 amino acids  =  36.0 kDa
Product: Renilla luciferase
human codon-optimized
AmpR
3105 .. 3965  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3105 .. 3896  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3105 .. 3965  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3897 .. 3965  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3105 .. 3965  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
HSV TK promoter
43 .. 794  =  752 bp
herpes simplex virus thymidine kinase promoter
HSV TK promoter
43 .. 794  =  752 bp
herpes simplex virus thymidine kinase promoter
ori
2346 .. 2934  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2346 .. 2934  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4097 .. 4552  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4097 .. 4552  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 poly(A) signal
1806 .. 1927  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1806 .. 1927  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3966 .. 4070  =  105 bp
AmpR promoter
3966 .. 4070  =  105 bp
pause site
4745 .. 4836  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
pause site
4745 .. 4836  =  92 bp
RNA polymerase II transcriptional pause signal from
the human α2 globin gene
poly(A) signal
4683 .. 4731  =  49 bp
synthetic polyadenylation signal
poly(A) signal
4683 .. 4731  =  49 bp
synthetic polyadenylation signal
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