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Plasmid Files

pdCas9-bacteria

Bacterial plasmid for tetracycline-inducible expression of catalytically dead S. pyogenes Cas9.

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pdCas9-bacteria Sequence and MappdCas9-bacteria.dna
Map and Sequence File   
Sequence Author:  Qi Lab / Addgene #44249
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 ZraI (3) BspEI (6365) PflMI * (6136) PasI (6130) MscI (6098) SpeI (5793) EcoP15I (5506) AlwNI (5484) AgeI (5291) BssS α I (5258) SacII (5133) PfoI * (5086) BsrBI (5055) AvrII (4991) T7Te terminator BmeT110I (4848) AvaI - BsoBI - PaeR7I - PspXI - XhoI (4847) BamHI (4117) MluI (3311) AatII (5) AflII (6) AfeI (225) HaeII (227) NsiI (244) SnaBI (392) EcoNI (420) BglII (716) RBS BstZ17I (954) BmgBI (959) SwaI (1260) BfuAI - BspMI (1808) NheI (1838) BmtI (1842) SphI (2000) Acc65I (2513) KpnI (2517) PmlI (3032) PciI (3201) pdCas9-bacteria 6705 bp
ZraI  (3)
1 site
G A C G T C C T G C A G
BspEI  (6365)
1 site
T C C G G A A G G C C T
PflMI  (6136)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
PasI  (6130)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
MscI  (6098)
1 site
T G G C C A A C C G G T
SpeI  (5793)
1 site
A C T A G T T G A T C A
EcoP15I  (5506)
1 site
C A G C A G ( N ) 25 G T C G T C ( N ) 25 N N

Efficient cleavage requires two inversely oriented copies of the
EcoP15I recognition sequence.
Sticky ends from different EcoP15I sites may not be compatible.
EcoP15I requires ATP for activity.
AlwNI  (5484)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
AgeI  (5291)
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.
BssSαI  (5258)
1 site
C A C G A G G T G C T C
SacII  (5133)
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.
PfoI  (5086)
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.
BsrBI  (5055)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures
up to 50°C.
AvrII  (4991)
1 site
C C T A G G G G A T C C
BmeT110I  (4848)
1 site
C Y C G R G G R G C Y C
AvaI  (4847)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (4847)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
PaeR7I  (4847)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4847)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4847)
1 site
C T C G A G G A G C T C
BamHI  (4117)
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.
MluI  (3311)
1 site
A C G C G T T G C G C A
AatII  (5)
1 site
G A C G T C C T G C A G
AflII  (6)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
AfeI  (225)
1 site
A G C G C T T C G C G A
HaeII  (227)
1 site
R G C G C Y Y C G C G R
NsiI  (244)
1 site
A T G C A T T A C G T A
SnaBI  (392)
1 site
T A C G T A A T G C A T
EcoNI  (420)
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.
BglII  (716)
1 site
A G A T C T T C T A G A
BstZ17I  (954)
1 site
G T A T A C C A T A T G
BmgBI  (959)
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.
SwaI  (1260)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BfuAI  (1808)
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  (1808)
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.
NheI  (1838)
1 site
G C T A G C C G A T C G
BmtI  (1842)
1 site
G C T A G C C G A T C G
SphI  (2000)
1 site
G C A T G C C G T A C G
Acc65I  (2513)
1 site
G G T A C C C C A T G G
KpnI  (2517)
1 site
G G T A C C C C A T G G
PmlI  (3032)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PciI  (3201)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
dCas9
740 .. 4846  =  4107 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   740 .. 766  =  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
740 .. 4846  =  4107 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   767 .. 769  =  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
740 .. 4846  =  4107 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   770 .. 3256  =  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
740 .. 4846  =  4107 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3257 .. 3259  =  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
740 .. 4846  =  4107 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3260 .. 4846  =  1587 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
740 .. 4846  =  4107 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
CmR
5920 .. 6579  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
5920 .. 6579  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
TetR
7 .. 630  =  624 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
7 .. 630  =  624 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.
p15A ori
5146 .. 5691  =  546 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
p15A ori
5146 .. 5691  =  546 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
cat promoter
6580 .. 6682  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
cat promoter
6580 .. 6682  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
lambda t0 terminator
5805 .. 5899  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
5805 .. 5899  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
4870 .. 4941  =  72 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
4870 .. 4941  =  72 bp
transcription terminator T1 from the E. coli rrnB
gene
tetR/tetA promoters
649 .. 704  =  56 bp
overlapping promoters for bacterial tetR and tetA
tetR/tetA promoters
649 .. 704  =  56 bp
overlapping promoters for bacterial tetR and tetA
T7Te terminator
4957 .. 4984  =  28 bp
phage T7 early transcription terminator
T7Te terminator
4957 .. 4984  =  28 bp
phage T7 early transcription terminator
RBS
722 .. 733  =  12 bp
strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)
RBS
722 .. 733  =  12 bp
strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)
tet operator
685 .. 703  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
685 .. 703  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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