pRSIT17-U6Tet-sh-HTS6-CMV-TetRep-2A-TagGFP2-2A-Puro

Lentiviral vector for inducible barcoded shRNA libraries, with an HTS6 cassette for high-throughput sequencing plus TagGFP2 and puromycin resistance markers.

Sequence Author: Cellecta

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MluI (1) SspI (8089) ScaI (7765) PciI (6392) SfiI (5970) AanI (5741) AanI (5685) KpnI (5350) Acc65I (5346) HincII (4751) SalI (4749) SexAI * (4710) EagI (4591) SphI (96) NruI * (612) MfeI (964) BbvCI (1199) 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) Bsu36I (2898) BlpI (3208) BclI * (3238) BsaBI * (3936) NheI (4065) BmtI (4069) PflFI - Tth111I (4185) BsiWI (4199) RsrII (4259) pRSIT17-U6Tet-sh-HTS6-CMV- TetRep-2A-TagGFP2-2A-Puro 8676 bp
MluI  (1)
1 site
A C G C G T T G C G C A
SspI  (8089)
1 site
A A T A T T T T A T A A
ScaI  (7765)
1 site
A G T A C T T C A T G A
PciI  (6392)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SfiI  (5970)
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  (5741)
2 sites
T T A T A A A A T A T T
AanI  (5685)
2 sites
T T A T A A A A T A T T
KpnI  (5350)
1 site
G G T A C C C C A T G G
Acc65I  (5346)
1 site
G G T A C C C C A T G G
HincII  (4751)
1 site
G T Y R A C C A R Y T G
SalI  (4749)
1 site
G T C G A C C A G C T G
SexAI  (4710)
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  (4591)
1 site
C G G C C G G C C G G 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
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.
Bsu36I  (2898)
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.
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.
BsaBI  (3936)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
NheI  (4065)
1 site
G C T A G C C G A T C G
BmtI  (4069)
1 site
G C T A G C C G A T C G
PflFI  (4185)
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  (4185)
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  (4199)
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  (4259)
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.
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.
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.
TagGFP2
3351 .. 4064  =  714 bp
238 amino acids  =  26.9 kDa
Product: monomeric green fluorescent protein, also known as mTagGFP
mammalian codon-optimized
TagGFP2
3351 .. 4064  =  714 bp
238 amino acids  =  26.9 kDa
Product: monomeric green fluorescent protein, also known as mTagGFP
mammalian codon-optimized
T2A
4083 .. 4136  =  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
4083 .. 4136  =  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.
PuroR
4143 .. 4742  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
4143 .. 4742  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
AmpR
7212 .. 8072  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   7212 .. 8003  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7212 .. 8072  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   8004 .. 8072  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
7212 .. 8072  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
WPRE
4755 .. 5343  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
WPRE
4755 .. 5343  =  589 bp
woodchuck hepatitis virus posttranscriptional regulatory element
ori
6453 .. 7041  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
6453 .. 7041  =  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)
5417 .. 5650  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from HIV-1
3' LTR (ΔU3)
5417 .. 5650  =  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
5883 .. 6018  =  136 bp
SV40 origin of replication
SV40 ori
5883 .. 6018  =  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
5722 .. 5843  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5722 .. 5843  =  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
8073 .. 8177  =  105 bp
AmpR promoter
8073 .. 8177  =  105 bp
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
ORF:  2908 .. 3240  =  333 bp
ORF:  110 amino acids  =  12.4 kDa
ORF:  4858 .. 5448  =  591 bp
ORF:  196 amino acids  =  20.9 kDa
ORF:  7342 .. 7608  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  8458 .. 180  =  399 bp
ORF:  132 amino acids  =  15.1 kDa
ORF:  956 .. 1837  =  882 bp
ORF:  293 amino acids  =  33.8 kDa
ORF:  5744 .. 5980  =  237 bp
ORF:  78 amino acids  =  8.6 kDa
ORF:  2664 .. 4742  =  2079 bp
ORF:  692 amino acids  =  76.4 kDa
ORF:  4845 .. 5390  =  546 bp
ORF:  181 amino acids  =  19.7 kDa
ORF:  871 .. 1251  =  381 bp
ORF:  126 amino acids  =  13.3 kDa
ORF:  8418 .. 161  =  420 bp
ORF:  139 amino acids  =  15.8 kDa
ORF:  7212 .. 8072  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2594 .. 2833  =  240 bp
ORF:  79 amino acids  =  8.7 kDa
ORF:  2960 .. 3382  =  423 bp
ORF:  140 amino acids  =  15.4 kDa
ORF:  3623 .. 3862  =  240 bp
ORF:  79 amino acids  =  9.4 kDa
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