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Plasmid Files

pwtCas9-bacteria

Bacterial plasmid for tetracycline-inducible expression of catalytically active wild-type S. pyogenes Cas9.

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pwtCas9-bacteria Sequence and MappwtCas9-bacteria.dna
Map and Sequence File   
Sequence Author:  Qi Lab / Addgene #44250
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 EaeI (6412) PvuI (6394) NmeAIII (6172) BglI (6144) BsrFI (6104) BsaI (6085) AhdI (6024) SpeI (5815) AlwNI (5506) PspFI (5398) BseYI (5394) AatII (5003) ZraI (5001) AvrII (4991) BmeT110I (4848) AvaI - BsoBI - PaeR7I - PspXI - XhoI (4847) BamHI (4117) AflII (6) AfeI (225) NsiI (244) SnaBI (392) EcoNI (420) XbaI (622) BglII (716) RBS BseRI (740) BstZ17I (954) BmgBI (959) SwaI (1260) BlpI (1419) BfuAI - BspMI (1808) NheI (1838) BmtI (1842) BstBI (2077) BtgI - NcoI (2162) Acc65I (2513) KpnI (2517) PvuII (2824) PmlI (3032) MluI (3311) pwtCas9-bacteria 6932 bp
EaeI  (6412)
1 site
Y G G C C R R C C G G Y
PvuI  (6394)
1 site
C G A T C G G C T A G C
NmeAIII  (6172)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BglI  (6144)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
BsrFI  (6104)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
BsaI  (6085)
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  (6024)
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.
SpeI  (5815)
1 site
A C T A G T T G A T C A
AlwNI  (5506)
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.
PspFI  (5398)
1 site
C C C A G C G G G T C G
BseYI  (5394)
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.
AatII  (5003)
1 site
G A C G T C C T G C A G
ZraI  (5001)
1 site
G A C G T C C T G C A G
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.
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
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.
XbaI  (622)
1 site
T C T A G A A G A T C T
BglII  (716)
1 site
A G A T C T T C T A G A
BseRI  (740)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
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.
BlpI  (1419)
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.
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
BstBI  (2077)
1 site
T T C G A A A A G C T T
BtgI  (2162)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (2162)
1 site
C C A T G G G G T A C C
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
PvuII  (2824)
1 site
C A G C T G G T C G A C
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.
MluI  (3311)
1 site
A C G C G T T G C G C A
Cas9
740 .. 4846  =  4107 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
740 .. 4846  =  4107 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
5951 .. 6811  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5951 .. 6742  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5951 .. 6811  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6743 .. 6811  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5951 .. 6811  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
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.
ori
5151 .. 5739  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5151 .. 5739  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
6812 .. 6916  =  105 bp
AmpR promoter
6812 .. 6916  =  105 bp
lambda t0 terminator
5827 .. 5921  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
5827 .. 5921  =  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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