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pRSITUR-U6Tet-sh-HTS3-UbiC-TetRep-2A-TagRFP

Lentiviral vector for inducible barcoded shRNA libraries, with an HTS3 cassette for high-throughput sequencing plus a TagRFP marker.

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 MluI (1) SspI (7517) ScaI (7193) PciI (5820) SfiI (5398) NcoI (5352) AanI (5169) AanI (5113) KpnI (4778) Acc65I (4774) NaeI (4697) NgoMIV (4695) HincII (4179) SalI (4177) SphI (96) BssHII (490) NruI * (612) MfeI (964) BbvCI (1199) AleI (1352) PpuMI - SanDI (1709) BspDI - ClaI (1798) BsaAI (1928) BbsI - BpiI (2065) cloning site BbsI - BpiI (2091) EcoRI (2154) PaeR7I - XhoI (2165) BstBI (2356) AfeI (2416) XbaI (2760) BstXI (2856) BlpI (3314) BclI * (3344) BamHI (3391) BspEI (3451) BsrGI (3506) PasI (3687) PshAI (4096) pRSITUR-U6Tet-sh-HTS3-UbiC-TetRep-2A-TagRFP 8104 bp
MluI  (1)
1 site
A C G C G T T G C G C A
SspI  (7517)
1 site
A A T A T T T T A T A A
ScaI  (7193)
1 site
A G T A C T T C A T G A
PciI  (5820)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SfiI  (5398)
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  (5352)
1 site
C C A T G G G G T A C C
AanI  (5169)
2 sites
T T A T A A A A T A T T
AanI  (5113)
2 sites
T T A T A A A A T A T T
KpnI  (4778)
1 site
G G T A C C C C A T G G
Acc65I  (4774)
1 site
G G T A C C C C A T G G
NaeI  (4697)
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.
NgoMIV  (4695)
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.
HincII  (4179)
1 site
G T Y R A C C A R Y T G
SalI  (4177)
1 site
G T C G A C C A G C T G
SphI  (96)
1 site
G C A T G C C G T A C G
BssHII  (490)
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  (612)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
MfeI  (964)
1 site
C A A T T G G T T A A C
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  (2154)
1 site
G A A T T C C T T A A G
PaeR7I  (2165)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2165)
1 site
C T C G A G G A G C T C
BstBI  (2356)
1 site
T T C G A A A A G C T T
AfeI  (2416)
1 site
A G C G C T T C G C G A
XbaI  (2760)
1 site
T C T A G A A G A T C T
BstXI  (2856)
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  (3314)
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  (3344)
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  (3391)
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.
BspEI  (3451)
1 site
T C C G G A A G G C C T
BsrGI  (3506)
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.
PasI  (3687)
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.
PshAI  (4096)
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.
AmpR
6640 .. 7500  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6640 .. 7431  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6640 .. 7500  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7432 .. 7500  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6640 .. 7500  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagRFP
3457 .. 4170  =  714 bp
237 amino acids  =  26.7 kDa
Product: monomeric derivative of red fluorescent
protein from Entacmaea quadricolor (Merzlyak et
al., 2007)
mammalian codon-optimized
TagRFP
3457 .. 4170  =  714 bp
237 amino acids  =  26.7 kDa
Product: monomeric derivative of red fluorescent
protein from Entacmaea quadricolor (Merzlyak et
al., 2007)
mammalian codon-optimized
TetR
2770 .. 3390  =  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
2770 .. 3390  =  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.
WPRE
4183 .. 4771  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
4183 .. 4771  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
5881 .. 6469  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5881 .. 6469  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
UbiC promoter
2361 .. 2760  =  400 bp
human ubiquitin C promoter
UbiC promoter
2361 .. 2760  =  400 bp
human ubiquitin C promoter
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)
4845 .. 5078  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
4845 .. 5078  =  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
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
5311 .. 5446  =  136 bp
SV40 origin of replication
SV40 ori
5311 .. 5446  =  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
5150 .. 5271  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5150 .. 5271  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2195 .. 2312  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2195 .. 2312  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
7501 .. 7605  =  105 bp
AmpR promoter
7501 .. 7605  =  105 bp
T2A
3397 .. 3450  =  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
3397 .. 3450  =  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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