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Plasmid Files

scramble shRNA

Third generation lentiviral negative control vector with a scrambled shRNA.

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scramble shRNA Sequence and Mapscramble shRNA.dna
Map and Sequence File   
Sequence Author:  Sabatini Lab / Addgene #1864
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 AscI (7067) BlpI (7058) SpeI (6978) XcmI (6750) EcoRI (6515) AgeI (6456) NdeI (6388) BspDI * - ClaI * (6170) KflI - PpuMI (6081) AleI (5724) BbvCI - Bpu10I (5571) MfeI (5336) EcoNI (5317) NruI * (4984) SphI (4468) AflIII - PciI (3965) PspFI (3665) HincII (120) BamHI (150) PflFI - Tth111I (211) BsiWI (225) RsrII (285) BstEII (303) SexAI * (736) NsiI (822) Acc65I (825) KpnI (829) BbsI (927) NcoI (1403) SfiI (1449) AvrII (1496) NgoMIV (1829) NaeI (1831) ScaI (2596) BseYI (3661) scramble shRNA 7085 bp
AscI  (7067)
1 site
G G C G C G C C C C G C G C G G
BlpI  (7058)
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.
SpeI  (6978)
1 site
A C T A G T T G A T C A
XcmI  (6750)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
EcoRI  (6515)
1 site
G A A T T C C T T A A G
AgeI  (6456)
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.
NdeI  (6388)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
BspDI  (6170)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (6170)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
KflI  (6081)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (6081)
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.
AleI  (5724)
1 site
C A C N N N N G T G G T G N N N N C A C
BbvCI  (5571)
1 site
C C T C A G C G G A G T C G
Bpu10I  (5571)
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.
MfeI  (5336)
1 site
C A A T T G G T T A A C
EcoNI  (5317)
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.
NruI  (4984)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
SphI  (4468)
1 site
G C A T G C C G T A C G
AflIII  (3965)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (3965)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (3665)
1 site
C C C A G C G G G T C G
HincII  (120)
1 site
G T Y R A C C A R Y T G
BamHI  (150)
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  (211)
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  (211)
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  (225)
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  (285)
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  (303)
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.
SexAI  (736)
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.
NsiI  (822)
1 site
A T G C A T T A C G T A
Acc65I  (825)
1 site
G G T A C C C C A T G G
KpnI  (829)
1 site
G G T A C C C C A T G G
BbsI  (927)
1 site
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.
NcoI  (1403)
1 site
C C A T G G G G T A C C
SfiI  (1449)
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.
AvrII  (1496)
1 site
C C T A G G G G A T C C
NgoMIV  (1829)
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.
NaeI  (1831)
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.
ScaI  (2596)
1 site
A G T A C T T C A T G A
BseYI  (3661)
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.
AmpR
2290 .. 3150  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2290 .. 2358  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2290 .. 3150  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2359 .. 3150  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2290 .. 3150  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
169 .. 768  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
169 .. 768  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
3321 .. 3909  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3321 .. 3909  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hPGK promoter
6722 .. 147  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
6722 .. 147  =  511 bp
human phosphoglycerate kinase 1 promoter
f1 ori
1704 .. 2159  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1704 .. 2159  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
U6 promoter
6210 .. 6450  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
6210 .. 6450  =  241 bp
RNA polymerase III promoter for human U6 snRNA
3' LTR (ΔU3)
896 .. 1129  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
896 .. 1129  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RRE
5450 .. 5683  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
5450 .. 5683  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RSV promoter
4378 .. 4604  =  227 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
4378 .. 4604  =  227 bp
Rous sarcoma virus enhancer/promoter
5' LTR (truncated)
4605 .. 4785  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
4605 .. 4785  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
1362 .. 1497  =  136 bp
SV40 origin of replication
SV40 ori
1362 .. 1497  =  136 bp
SV40 origin of replication
HIV-1 Ψ
4832 .. 4957  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
4832 .. 4957  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
1201 .. 1322  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1201 .. 1322  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
6556 .. 6673  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
6556 .. 6673  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
2185 .. 2289  =  105 bp
AmpR promoter
2185 .. 2289  =  105 bp
shRNA
6462 .. 6509  =  48 bp
shRNA
6462 .. 6509  =  48 bp
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