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Plasmid Files

pMLM3705

CRISPR activation plasmid for mammalian cell expression of catalytically inactive dCas9 fused to the VP64 transcriptional activation domain.

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pMLM3705 Sequence and MappMLM3705.dna
Map and Sequence File   
Sequence Author:  Joung Lab / Addgene #47754
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 CMV enhancer SpeI (7551) MluI (7530) SgrDI (7296) ScaI (6855) PvuI (6745) BsaI (6436) AhdI (6375) BspQI - SapI (5366) BbsI (5035) PmeI (4824) AgeI (4794) AscI - BssHII (4635) PstI (4617) PaeR7I - XhoI (4539) SV40 NLS BamHI (4513) BmtI (4055) EagI - NotI - SacII (370) BstZ17I (623) BmgBI (628) MscI (679) Bsu36I (859) BstEII (1123) StuI (1148) BsiWI (1217) KasI (1350) NarI (1351) SfoI (1352) PluTI (1354) BfuAI - BspMI (1477) BsrGI (1967) PfoI * (2225) EcoRV (2250) PshAI (2391) FspAI (3359) BsgI (3727) NheI (4051) pMLM3705 7785 bp
SpeI  (7551)
1 site
A C T A G T T G A T C A
MluI  (7530)
1 site
A C G C G T T G C G C A
SgrDI  (7296)
1 site
C G T C G A C G G C A G C T G C
ScaI  (6855)
1 site
A G T A C T T C A T G A
PvuI  (6745)
1 site
C G A T C G G C T A G C
BsaI  (6436)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (6375)
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.
BspQI  (5366)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (5366)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BbsI  (5035)
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.
PmeI  (4824)
1 site
G T T T A A A C C A A A T T T G
AgeI  (4794)
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.
AscI  (4635)
1 site
G G C G C G C C C C G C G C G G
BssHII  (4635)
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.
PstI  (4617)
1 site
C T G C A G G A C G T C
PaeR7I  (4539)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (4539)
1 site
C T C G A G G A G C T C
BamHI  (4513)
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.
BmtI  (4055)
1 site
G C T A G C C G A T C G
EagI  (370)
1 site
C G G C C G G C C G G C
NotI  (370)
1 site
G C G G C C G C C G C C G G C G
SacII  (370)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BstZ17I  (623)
1 site
G T A T A C C A T A T G
BmgBI  (628)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
MscI  (679)
1 site
T G G C C A A C C G G T
Bsu36I  (859)
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.
BstEII  (1123)
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.
StuI  (1148)
1 site
A G G C C T T C C G G A
BsiWI  (1217)
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.
KasI  (1350)
1 site
G G C G C C C C G C G G
NarI  (1351)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (1352)
1 site
G G C G C C C C G C G G
PluTI  (1354)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
BfuAI  (1477)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1477)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsrGI  (1967)
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.
PfoI  (2225)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
EcoRV  (2250)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
PshAI  (2391)
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.
FspAI  (3359)
1 site
R T G C G C A Y Y A C G C G T R
BsgI  (3727)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
NheI  (4051)
1 site
G C T A G C C G A T C G
dCas9
409 .. 4512  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   409 .. 435  =  27 bp
   9 amino acids  =  1.1 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
409 .. 4512  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   436 .. 438  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
409 .. 4512  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   439 .. 2925  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
409 .. 4512  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   2926 .. 2928  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
409 .. 4512  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   2929 .. 4512  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
409 .. 4512  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
AmpR
6302 .. 7162  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6302 .. 7093  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6302 .. 7162  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7094 .. 7162  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6302 .. 7162  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
5543 .. 6131  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5543 .. 6131  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
7537 .. 131  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
7537 .. 131  =  380 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
4846 .. 5070  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
4846 .. 5070  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
132 .. 335  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
132 .. 335  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
VP64
4642 .. 4791  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
VP64
4642 .. 4791  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
AmpR promoter
7163 .. 7267  =  105 bp
AmpR promoter
7163 .. 7267  =  105 bp
3xFLAG
4546 .. 4611  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
4546 .. 4611  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
SV40 NLS
4519 .. 4539  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4519 .. 4539  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
377 .. 395  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
377 .. 395  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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