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Plasmid Files

pRetroX-PTuner

Retroviral vector for fusing a destabilization domain to the N-terminus of a partner protein.

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pRetroX-PTuner Sequence and MappRetroX-PTuner.dna
Map and Sequence File   
Sequence Author:  Clontech
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 NdeI (6006) SspI (5639) ScaI (5315) BspQI - SapI (3826) AgeI - SgrAI (2933) AscI (446) SpeI (729) PshAI (851) PstI (1014) MluI (1068) BtgZI (1138) EF-1 α intron A BsgI (1395) NotI (1695) BglII (1702) BspDI - ClaI (1709) BamHI (1714) NcoI (1722) AvrII (1881) PflMI (2183) BmgBI (2273) BsiWI (2392) SalI (2444) AccI (2445) HincII (2446) RsrII (2452) BstEII (2470) StuI (2667) SexAI * (2903) pRetroX-PTuner 6229 bp
NdeI  (6006)
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.
SspI  (5639)
1 site
A A T A T T T T A T A A
ScaI  (5315)
1 site
A G T A C T T C A T G A
BspQI  (3826)
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  (3826)
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.
AgeI  (2933)
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  (2933)
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.
AscI  (446)
1 site
G G C G C G C C C C G C G C G G
SpeI  (729)
1 site
A C T A G T T G A T C A
PshAI  (851)
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.
PstI  (1014)
1 site
C T G C A G G A C G T C
MluI  (1068)
1 site
A C G C G T T G C G C A
BtgZI  (1138)
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.
BsgI  (1395)
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  (1695)
1 site
G C G G C C G C C G C C G G C G
BglII  (1702)
1 site
A G A T C T T C T A G A
BspDI  (1709)
1 site
A T C G A T T A G C T A
ClaI  (1709)
1 site
A T C G A T T A G C T A
BamHI  (1714)
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.
NcoI  (1722)
1 site
C C A T G G G G T A C C
AvrII  (1881)
1 site
C C T A G G G G A T C C
PflMI  (2183)
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.
BmgBI  (2273)
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.
BsiWI  (2392)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SalI  (2444)
1 site
G T C G A C C A G C T G
AccI  (2445)
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.
HincII  (2446)
1 site
G T Y R A C C A R Y T G
RsrII  (2452)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BstEII  (2470)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
StuI  (2667)
1 site
A G G C C T T C C G G A
SexAI  (2903)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
AmpR
4762 .. 5622  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4762 .. 5553  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4762 .. 5622  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5554 .. 5622  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4762 .. 5622  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
2336 .. 2932  =  597 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
2336 .. 2932  =  597 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
LTR
2992 .. 3585  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
LTR
2992 .. 3585  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
LTR
1 .. 592  =  592 bp
long terminal repeat from Moloney murine leukemia
virus
LTR
1 .. 592  =  592 bp
long terminal repeat from Moloney murine leukemia
virus
ori
4003 .. 4591  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4003 .. 4591  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
1730 .. 2303  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
1730 .. 2303  =  574 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
MMLV Ψ
655 .. 1012  =  358 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
MMLV Ψ
655 .. 1012  =  358 bp
packaging signal of Moloney murine leukemia virus
(MMLV)
DD
1368 .. 1691  =  324 bp
108 amino acids  =  11.9 kDa
   Segment 1:  
   1368 .. 1370  =  3 bp
   1 amino acid  =  149.2 Da
Product: destabilization domain that can be
stabilized by Shield1 in the ProteoTuner™ system
L106P mutant of FKBP12
DD
1368 .. 1691  =  324 bp
108 amino acids  =  11.9 kDa
   Segment 2:  
   1371 .. 1691  =  321 bp
   107 amino acids  =  11.8 kDa
Product: destabilization domain that can be
stabilized by Shield1 in the ProteoTuner™ system
L106P mutant of FKBP12
DD
1368 .. 1691  =  324 bp
108 amino acids  =  11.9 kDa
2 segments
Product: destabilization domain that can be
stabilized by Shield1 in the ProteoTuner™ system
L106P mutant of FKBP12
EF-1α intron A
1075 .. 1278  =  204 bp
truncated intron upstream of the start codon of
human EF-1α
EF-1α intron A
1075 .. 1278  =  204 bp
truncated intron upstream of the start codon of
human EF-1α
AmpR promoter
5623 .. 5727  =  105 bp
AmpR promoter
5623 .. 5727  =  105 bp
MCS
1694 .. 1727  =  34 bp
multiple cloning site
MCS
1694 .. 1727  =  34 bp
multiple cloning site
EF-1α exon
1279 .. 1308  =  30 bp
truncated exon 2 from human EF-1α, beginning with
a GT-to-CC mutation that reduces splicing efficiency
EF-1α exon
1279 .. 1308  =  30 bp
truncated exon 2 from human EF-1α, beginning with
a GT-to-CC mutation that reduces splicing efficiency
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