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pRSITEP-U6Tet-sh-noHTS-EF1-TetRep-2A-Puro

Lentiviral expression vector for inducible shRNA expression, with a puromycin resistance marker controlled by the EF-1α promoter.

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 MluI (1) SspI (7503) ScaI (7179) PciI (5806) SfiI (5384) NcoI (5338) AanI (5155) AanI (5099) KpnI (4764) Acc65I (4760) HincII (4165) SalI (4163) DraIII (4128) SphI (96) NruI * (612) BbvCI (1199) AleI (1352) PpuMI - SanDI (1709) BspDI - ClaI (1798) BsaAI (1928) BbsI - BpiI (2065) cloning site BbsI - BpiI (2091) EcoRI (2138) PaeR7I - XhoI (2149) SpeI (2339) 5' LTR (truncated) XbaI (2860) BstXI (2956) BlpI (3414) BclI * (3444) BamHI (3491) BmgBI (3529) PflFI - Tth111I (3599) PasI (3606) BsiWI (3613) BspEI * (3670) RsrII (3673) BstEII (3691) EagI (4005) SexAI * (4124) pRSITEP-U6Tet-sh-noHTS-EF1-TetRep-2A-Puro 8090 bp
MluI  (1)
1 site
A C G C G T T G C G C A
SspI  (7503)
1 site
A A T A T T T T A T A A
ScaI  (7179)
1 site
A G T A C T T C A T G A
PciI  (5806)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SfiI  (5384)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
NcoI  (5338)
1 site
C C A T G G G G T A C C
AanI  (5155)
2 sites
T T A T A A A A T A T T
AanI  (5099)
2 sites
T T A T A A A A T A T T
KpnI  (4764)
1 site
G G T A C C C C A T G G
Acc65I  (4760)
1 site
G G T A C C C C A T G G
HincII  (4165)
1 site
G T Y R A C C A R Y T G
SalI  (4163)
1 site
G T C G A C C A G C T G
DraIII  (4128)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
SphI  (96)
1 site
G C A T G C C G T A C G
NruI  (612)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BbvCI  (1199)
1 site
C C T C A G C G G A G T C G
AleI  (1352)
1 site
C A C N N N N G T G G T G N N N N C A C
PpuMI  (1709)
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.
SanDI  (1709)
1 site
G G G W C C C C C C W G G G

Sticky ends from different SanDI sites may not be compatible.
BspDI  (1798)
1 site
A T C G A T T A G C T A
ClaI  (1798)
1 site
A T C G A T T A G C T A
BsaAI  (1928)
1 site
Y A C G T R R T G C A Y
BbsI  (2065)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BpiI  (2065)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
BbsI  (2091)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BpiI  (2091)
2 sites
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BpiI sites may not be compatible.
EcoRI  (2138)
1 site
G A A T T C C T T A A G
PaeR7I  (2149)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2149)
1 site
C T C G A G G A G C T C
SpeI  (2339)
1 site
A C T A G T T G A T C A
XbaI  (2860)
1 site
T C T A G A A G A T C T
BstXI  (2956)
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.
BlpI  (3414)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BclI  (3444)
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.
BamHI  (3491)
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.
BmgBI  (3529)
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.
PflFI  (3599)
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  (3599)
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.
PasI  (3606)
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.
BsiWI  (3613)
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.
BspEI  (3670)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
RsrII  (3673)
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  (3691)
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.
EagI  (4005)
1 site
C G G C C G G C C G G C
SexAI  (4124)
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
6626 .. 7486  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6626 .. 7417  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6626 .. 7486  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7418 .. 7486  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6626 .. 7486  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TetR
2870 .. 3490  =  621 bp
207 amino acids  =  23.4 kDa
Product: tetracycline repressor TetR
TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved
by adding tetracycline or doxycycline.
TetR
2870 .. 3490  =  621 bp
207 amino acids  =  23.4 kDa
Product: tetracycline repressor TetR
TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved
by adding tetracycline or doxycycline.
PuroR
3557 .. 4156  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
3557 .. 4156  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
4169 .. 4757  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
4169 .. 4757  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
5867 .. 6455  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5867 .. 6455  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
5' LTR (truncated)
2569 .. 2837  =  269 bp
truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1
5' LTR (truncated)
2569 .. 2837  =  269 bp
truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1
U6-Tet promoter
1819 .. 2064  =  246 bp
RNA polymerase III promoter for human U6 snRNA,
modified to contain two tetracycline operator (tetO)
sites
U6-Tet promoter
1819 .. 2064  =  246 bp
RNA polymerase III promoter for human U6 snRNA,
modified to contain two tetracycline operator (tetO)
sites
RRE
1078 .. 1311  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1078 .. 1311  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
4831 .. 5064  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
4831 .. 5064  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RSV promoter
6 .. 232  =  227 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
6 .. 232  =  227 bp
Rous sarcoma virus enhancer/promoter
EF-1α core promoter
2345 .. 2556  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
2345 .. 2556  =  212 bp
core promoter for human elongation factor EF-1α
5' LTR (truncated)
233 .. 413  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
233 .. 413  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
5297 .. 5432  =  136 bp
SV40 origin of replication
SV40 ori
5297 .. 5432  =  136 bp
SV40 origin of replication
HIV-1 Ψ
460 .. 585  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
460 .. 585  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
5136 .. 5257  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5136 .. 5257  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2179 .. 2296  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2179 .. 2296  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
7487 .. 7591  =  105 bp
AmpR promoter
7487 .. 7591  =  105 bp
T2A
3497 .. 3550  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
3497 .. 3550  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
cloning site
2066 .. 2095  =  30 bp
shRNA cloning site
cloning site
2066 .. 2095  =  30 bp
shRNA cloning site
tet operator
2046 .. 2064  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2046 .. 2064  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2020 .. 2037  =  18 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
2020 .. 2037  =  18 bp
bacterial operator O2 for the tetR and tetA genes
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