pLVX-EF1alpha-IRES-ZsGreen1

Lentiviral bicistronic expression vector for constitutively co-expressing a protein of interest together with ZsGreen1 from the EF-1α promoter.

Sequence Author: Clontech (TaKaRa)

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SgrDI (8569) SspI (8452) PvuI (8018) FspI (7870) DrdI (6863) BmtI (6372) NheI (6368) Bsu36I (5578) NcoI (5266) PfoI (5131) MluI (4845) BsrGI (4699) PflFI - Tth111I (4383) BssHII (711) NruI * (833) KflI - PpuMI (1934) BspDI - ClaI (2180) SphI (2331) AgeI (2415) BstXI (2648) EcoRI (3535) SpeI (3547) XbaI (3553) NotI (3560) BamHI (3567) AvrII (3726) PaqCI (3914) BclI * (4231) SgrAI (4236) pLVX-EF1 α- IRES-ZsGreen1 8889 bp
SgrDI  (8569)
1 site
C G T C G A C G G C A G C T G C
SspI  (8452)
1 site
A A T A T T T T A T A A
PvuI  (8018)
1 site
C G A T C G G C T A G C
FspI  (7870)
1 site
T G C G C A A C G C G T
DrdI  (6863)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BmtI  (6372)
1 site
G C T A G C C G A T C G
NheI  (6368)
1 site
G C T A G C C G A T C G
Bsu36I  (5578)
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.
NcoI  (5266)
1 site
C C A T G G G G T A C C
PfoI  (5131)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
MluI  (4845)
1 site
A C G C G T T G C G C A
BsrGI  (4699)
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.
PflFI  (4383)
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  (4383)
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.
BssHII  (711)
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.
NruI  (833)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
KflI  (1934)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (1934)
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.
BspDI  (2180)
1 site
A T C G A T T A G C T A
ClaI  (2180)
1 site
A T C G A T T A G C T A
SphI  (2331)
1 site
G C A T G C C G T A C G
AgeI  (2415)
1 site
A C C G G T T G G C C A
BstXI  (2648)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
EcoRI  (3535)
1 site
G A A T T C C T T A A G
SpeI  (3547)
1 site
A C T A G T T G A T C A
XbaI  (3553)
1 site
T C T A G A A G A T C T
NotI  (3560)
1 site
G C G G C C G C C G C C G G C G
BamHI  (3567)
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  (3726)
1 site
C C T A G G G G A T C C
PaqCI  (3914)
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 PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
BclI  (4231)
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  (4236)
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.
EF-1α promoter
2338 .. 3519  =  1182 bp
strong constitutive promoter for human elongation factor EF-1α
EF-1α promoter
2338 .. 3519  =  1182 bp
strong constitutive promoter for human elongation factor EF-1α
AmpR
7575 .. 8435  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   7575 .. 8366  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7575 .. 8435  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   8367 .. 8435  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7575 .. 8435  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ZsGreen1
4149 .. 4844  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
4149 .. 4844  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
3' LTR
1 .. 634  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
1 .. 634  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
5653 .. 6286  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
5653 .. 6286  =  634 bp
3' long terminal repeat (LTR) from HIV-1
WPRE
4858 .. 5446  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
4858 .. 5446  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
6816 .. 7404  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6816 .. 7404  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
3575 .. 4147  =  573 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
3575 .. 4147  =  573 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm.
RRE
1303 .. 1536  =  234 bp
The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm.
SV40 poly(A) signal
8588 .. 8722  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
8588 .. 8722  =  135 bp
SV40 polyadenylation signal
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus type 1
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus type 1
cPPT/CTS
2028 .. 2143  =  116 bp
central polypurine tract and central termination sequence of HIV-1 (lacking the first T)
cPPT/CTS
2028 .. 2143  =  116 bp
central polypurine tract and central termination sequence of HIV-1 (lacking the first T)
AmpR promoter
8436 .. 8540  =  105 bp
AmpR promoter
8436 .. 8540  =  105 bp
MCS
3535 .. 3572  =  38 bp
multiple cloning site
MCS
3535 .. 3572  =  38 bp
multiple cloning site
EF-1α intron A
2568 .. 3510  =  943 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
2568 .. 3510  =  943 bp
intron upstream of the start codon of human EF-1α
ORF:  4948 .. 5475  =  528 bp
ORF:  175 amino acids  =  19.0 kDa
ORF:  7705 .. 7971  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1181 .. 2032  =  852 bp
ORF:  283 amino acids  =  32.7 kDa
ORF:  2897 .. 3334  =  438 bp
ORF:  145 amino acids  =  15.9 kDa
ORF:  4961 .. 5257  =  297 bp
ORF:  98 amino acids  =  10.2 kDa
ORF:  2976 .. 3209  =  234 bp
ORF:  77 amino acids  =  7.7 kDa
ORF:  4149 .. 4844  =  696 bp
ORF:  231 amino acids  =  26.1 kDa
ORF:  1015 .. 1476  =  462 bp
ORF:  153 amino acids  =  16.6 kDa
ORF:  7575 .. 8435  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2612 .. 3259  =  648 bp
ORF:  215 amino acids  =  23.7 kDa
ORF:  4526 .. 4819  =  294 bp
ORF:  97 amino acids  =  10.9 kDa
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