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Plasmid Files

pRetroX-IRES-ZsGreen1

Bicistronic retroviral vector for expressing a gene together with the fluorescent protein ZsGreen1.

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pRetroX-IRES-ZsGreen1 Sequence and MappRetroX-IRES-ZsGreen1.dna
Map and Sequence File   
Sequence Author:  Clontech
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 NdeI (5730) SspI (5363) ScaI (5039) PspFI (3974) BseYI (3970) BspQI - SapI (3550) AscI (446) MscI (661) SpeI (729) PshAI (851) PstI (1014) MluI (1068) EF-1 α intron A NotI - SacII (1365) BglII (1372) BspDI - ClaI (1379) BamHI (1384) AvrII (1544) AarI (1732) PflMI (1846) BclI * (2049) SgrAI (2054) SalI (2204) AccI (2205) HincII (2206) NgoMIV (2231) NaeI (2233) BsrGI (2517) pRetroX-IRES-ZsGreen1 5953 bp
NdeI  (5730)
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  (5363)
1 site
A A T A T T T T A T A A
ScaI  (5039)
1 site
A G T A C T T C A T G A
PspFI  (3974)
1 site
C C C A G C G G G T C G
BseYI  (3970)
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.
BspQI  (3550)
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  (3550)
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
MscI  (661)
1 site
T G G C C A A C C G G T
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
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.
PflMI  (1846)
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.
BclI  (2049)
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.
SgrAI  (2054)
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.
SalI  (2204)
1 site
G T C G A C C A G C T G
AccI  (2205)
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  (2206)
1 site
G T Y R A C C A R Y T G
NgoMIV  (2231)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (2233)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
BsrGI  (2517)
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.
AmpR
4486 .. 5346  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4486 .. 5277  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4486 .. 5346  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5278 .. 5346  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4486 .. 5346  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ZsGreen1
1967 .. 2662  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
1967 .. 2662  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
3' LTR
2716 .. 3309  =  594 bp
long terminal repeat from Moloney murine leukemia
virus
3' LTR
2716 .. 3309  =  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
3727 .. 4315  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3727 .. 4315  =  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
5347 .. 5451  =  105 bp
AmpR promoter
5347 .. 5451  =  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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