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pRSI17cb-U6-sh-13kCB18-HTS6-UbiC-TagGFP2-2A-Puro

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

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 MluI (1) SspI (7463) ScaI (7139) AhdI (6659) PspFI (6074) BseYI (6070) PciI (5766) SfiI (5344) NcoI (5298) AanI (5115) AanI (5059) KpnI (4724) Acc65I (4720) HincII (4125) SalI (4123) SexAI * (4084) SphI (96) NruI * (612) EcoNI (945) MfeI (964) BbvCI (1199) PpuMI - SanDI (1709) BspDI - ClaI (1798) BsaAI (1928) BbsI - BpiI (2064) cloning site BbsI - BpiI (2090) PaeR7I - PspXI - XhoI (2135) EcoRI (2302) AgeI (2311) BstBI (2320) AfeI (2380) XbaI (2724) BsaBI * (3322) BamHI (3451) PflFI - Tth111I (3559) BsiWI (3573) BspEI * (3630) RsrII (3633) MscI * (3892) EagI (3965) pRSI17cb-U6-sh-13kCB18-HTS6-UbiC-TagGFP2-2A-Puro 8050 bp
MluI  (1)
1 site
A C G C G T T G C G C A
SspI  (7463)
1 site
A A T A T T T T A T A A
ScaI  (7139)
1 site
A G T A C T T C A T G A
AhdI  (6659)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
PspFI  (6074)
1 site
C C C A G C G G G T C G
BseYI  (6070)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
PciI  (5766)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SfiI  (5344)
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  (5298)
1 site
C C A T G G G G T A C C
AanI  (5115)
2 sites
T T A T A A A A T A T T
AanI  (5059)
2 sites
T T A T A A A A T A T T
KpnI  (4724)
1 site
G G T A C C C C A T G G
Acc65I  (4720)
1 site
G G T A C C C C A T G G
HincII  (4125)
1 site
G T Y R A C C A R Y T G
SalI  (4123)
1 site
G T C G A C C A G C T G
SexAI  (4084)
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.
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.
EcoNI  (945)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
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
BsaAI  (1928)
1 site
Y A C G T R R T G C A Y
BbsI  (2064)
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  (2064)
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  (2090)
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  (2090)
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  (2135)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2135)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2135)
1 site
C T C G A G G A G C T C
EcoRI  (2302)
1 site
G A A T T C C T T A A G
AgeI  (2311)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BstBI  (2320)
1 site
T T C G A A A A G C T T
AfeI  (2380)
1 site
A G C G C T T C G C G A
XbaI  (2724)
1 site
T C T A G A A G A T C T
BsaBI  (3322)
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.
BamHI  (3451)
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.
PflFI  (3559)
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  (3559)
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  (3573)
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  (3630)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
RsrII  (3633)
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.
MscI  (3892)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
EagI  (3965)
1 site
C G G C C G G C C G G C
AmpR
6586 .. 7446  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6586 .. 7377  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6586 .. 7446  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7378 .. 7446  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6586 .. 7446  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagGFP2
2737 .. 3450  =  714 bp
238 amino acids  =  26.9 kDa
Product: monomeric green fluorescent protein, also
known as mTagGFP
mammalian codon-optimized
TagGFP2
2737 .. 3450  =  714 bp
238 amino acids  =  26.9 kDa
Product: monomeric green fluorescent protein, also
known as mTagGFP
mammalian codon-optimized
PuroR
3517 .. 4116  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
3517 .. 4116  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
4129 .. 4717  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
4129 .. 4717  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
5827 .. 6415  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5827 .. 6415  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
UbiC promoter
2325 .. 2724  =  400 bp
human ubiquitin C promoter
UbiC promoter
2325 .. 2724  =  400 bp
human ubiquitin C promoter
U6 promoter
1819 .. 2059  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
1819 .. 2059  =  241 bp
RNA polymerase III promoter for human U6 snRNA
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)
4791 .. 5024  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
4791 .. 5024  =  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
5257 .. 5392  =  136 bp
SV40 origin of replication
SV40 ori
5257 .. 5392  =  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
5096 .. 5217  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5096 .. 5217  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2144 .. 2259  =  116 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2144 .. 2259  =  116 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
7447 .. 7551  =  105 bp
AmpR promoter
7447 .. 7551  =  105 bp
T2A
3457 .. 3510  =  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
3457 .. 3510  =  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
2065 .. 2094  =  30 bp
shRNA cloning site
cloning site
2065 .. 2094  =  30 bp
shRNA cloning site
clonal barcode
2095 .. 2112  =  18 bp
clonal barcode
2095 .. 2112  =  18 bp
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