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Plasmid Files

pRetroX-IRES-DsRedExpress

Bicistronic retroviral vector for expressing a gene together with the fluorescent protein DsRed-Express.

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pRetroX-IRES-DsRedExpress Sequence and MappRetroX-IRES-DsRedExpress.dna
Map and Sequence File   
Sequence Author:  Clontech
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 NdeI (5711) SspI (5344) ScaI (5020) BspQI - SapI (3531) AscI (446) SpeI (729) PshAI (851) MluI (1068) BtgZI (1138) EF-1 α intron A NotI - SacII (1365) BglII (1372) BspDI - ClaI (1379) BamHI (1384) AvrII (1544) AarI - BfuAI - BspMI (1732) BmgBI (1935) FspAI (2015) SbfI (2307) StuI (2399) SexAI * (2484) BsgI (2546) AleI (2594) PaeR7I - PspXI - XhoI (2649) pRetroX-IRES-DsRedExpress 5934 bp
NdeI  (5711)
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  (5344)
1 site
A A T A T T T T A T A A
ScaI  (5020)
1 site
A G T A C T T C A T G A
BspQI  (3531)
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  (3531)
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.
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.
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.
NotI  (1365)
1 site
G C G G C C G C C G C C G G C G
SacII  (1365)
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.
BglII  (1372)
1 site
A G A T C T T C T A G A
BspDI  (1379)
1 site
A T C G A T T A G C T A
ClaI  (1379)
1 site
A T C G A T T A G C T A
BamHI  (1384)
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.
AvrII  (1544)
1 site
C C T A G G G G A T C C
AarI  (1732)
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  (1732)
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  (1732)
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.
BmgBI  (1935)
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.
FspAI  (2015)
1 site
R T G C G C A Y Y A C G C G T R
SbfI  (2307)
1 site
C C T G C A G G G G A C G T C C
StuI  (2399)
1 site
A G G C C T T C C G G A
SexAI  (2484)
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.
BsgI  (2546)
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).
AleI  (2594)
1 site
C A C N N N N G T G G T G N N N N C A C
PaeR7I  (2649)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2649)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2649)
1 site
C T C G A G G A G C T C
AmpR
4467 .. 5327  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4467 .. 5258  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4467 .. 5327  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5259 .. 5327  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4467 .. 5327  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
DsRed-Express
1967 .. 2644  =  678 bp
225 amino acids  =  25.7 kDa
Product: rapidly maturing tetrameric variant of
DsRed fluorescent protein
mammalian codon-optimized
DsRed-Express
1967 .. 2644  =  678 bp
225 amino acids  =  25.7 kDa
Product: rapidly maturing tetrameric variant of
DsRed fluorescent protein
mammalian codon-optimized
3' LTR
2697 .. 3290  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
3' LTR
2697 .. 3290  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
5' LTR
1 .. 592  =  592 bp
long terminal repeat from Moloney murine leukemia
virus
5' LTR
1 .. 592  =  592 bp
long terminal repeat from Moloney murine leukemia
virus
ori
3708 .. 4296  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3708 .. 4296  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
1393 .. 1965  =  573 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
1393 .. 1965  =  573 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)
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
5328 .. 5432  =  105 bp
AmpR promoter
5328 .. 5432  =  105 bp
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
MCS
1362 .. 1389  =  28 bp
multiple cloning site
MCS
1362 .. 1389  =  28 bp
multiple cloning site
splice donor
653 .. 658  =  6 bp
splice donor site (beginning of intron)
splice donor
653 .. 658  =  6 bp
splice donor site (beginning of intron)
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