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pRSIT16-U6Tet-sh-HTS6-CMV-TetRep-2A-TagRFP-2A-Puro

Lentiviral vector for inducible barcoded shRNA libraries, with an HTS6 cassette for high-throughput sequencing plus TagRFP and puromycin resistance markers.

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 MluI (1) SspI (8086) ScaI (7762) PciI (6389) SfiI (5967) AanI (5738) AanI (5682) KpnI (5347) Acc65I (5343) NaeI (5266) NgoMIV (5264) HincII (4748) SalI (4746) SexAI * (4707) EagI (4588) BstEII (4274) SphI (96) NruI * (612) MfeI (964) BbvCI - Bpu10I (1199) AleI (1352) PpuMI - SanDI (1709) BspDI - ClaI (1798) BbsI - BpiI (2065) cloning site BbsI - BpiI (2091) PaeR7I - PspXI - XhoI (2114) EcoRI (2281) SnaBI (2362) XbaI * (2654) BstXI (2750) BlpI (3208) BclI * (3238) BsrGI (3400) PshAI (3990) NheI (4062) BmtI (4066) PflFI - Tth111I (4182) BsiWI (4196) RsrII (4256) pRSIT16-U6Tet-sh-HTS6-CMV-TetRep-2A-TagRFP-2A-Puro 8673 bp
MluI  (1)
1 site
A C G C G T T G C G C A
SspI  (8086)
1 site
A A T A T T T T A T A A
ScaI  (7762)
1 site
A G T A C T T C A T G A
PciI  (6389)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SfiI  (5967)
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.
AanI  (5738)
2 sites
T T A T A A A A T A T T
AanI  (5682)
2 sites
T T A T A A A A T A T T
KpnI  (5347)
1 site
G G T A C C C C A T G G
Acc65I  (5343)
1 site
G G T A C C C C A T G G
NaeI  (5266)
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  (5264)
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  (4748)
1 site
G T Y R A C C A R Y T G
SalI  (4746)
1 site
G T C G A C C A G C T G
SexAI  (4707)
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.
EagI  (4588)
1 site
C G G C C G G C C G G C
BstEII  (4274)
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.
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.
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
Bpu10I  (1199)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
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
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.
PaeR7I  (2114)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2114)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2114)
1 site
C T C G A G G A G C T C
EcoRI  (2281)
1 site
G A A T T C C T T A A G
SnaBI  (2362)
1 site
T A C G T A A T G C A T
XbaI  (2654)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
BstXI  (2750)
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  (3208)
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  (3238)
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.
BsrGI  (3400)
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.
PshAI  (3990)
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.
NheI  (4062)
1 site
G C T A G C C G A T C G
BmtI  (4066)
1 site
G C T A G C C G A T C G
PflFI  (4182)
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  (4182)
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.
BsiWI  (4196)
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.
RsrII  (4256)
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.
AmpR
7209 .. 8069  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7209 .. 8000  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7209 .. 8069  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8001 .. 8069  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7209 .. 8069  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagRFP
3351 .. 4061  =  711 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
3351 .. 4061  =  711 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
2664 .. 3284  =  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
2664 .. 3284  =  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
4140 .. 4739  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
4140 .. 4739  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
4752 .. 5340  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
4752 .. 5340  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
6450 .. 7038  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6450 .. 7038  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
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)
5414 .. 5647  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
5414 .. 5647  =  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
CMV promoter
2387 .. 2590  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2387 .. 2590  =  204 bp
human cytomegalovirus (CMV) immediate early
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
5880 .. 6015  =  136 bp
SV40 origin of replication
SV40 ori
5880 .. 6015  =  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
5719 .. 5840  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5719 .. 5840  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2123 .. 2238  =  116 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2123 .. 2238  =  116 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
8070 .. 8174  =  105 bp
AmpR promoter
8070 .. 8174  =  105 bp
T2A
3291 .. 3344  =  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
3291 .. 3344  =  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
4080 .. 4133  =  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
4080 .. 4133  =  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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