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Plasmid Files

pCW-Cas9

Lentiviral vector for doxycycline-inducible expression of FLAG®-tagged Cas9.

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pCW-Cas9 Sequence and MappCW-Cas9.dna
Map and Sequence File   
Sequence Author:  Sabatini & Lander Labs / Addgene #50661
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 PaeR7I - XhoI (11) AleI (11,525) BbvCI (11,372) NotI (11,096) AflIII - PciI (9766) DrdI (9664) SspI (8073) AmpR promoter SfiI (7971) KpnI (7351) Acc65I (7347) SmaI (6738) TspMI - XmaI (6736) NdeI (6577) XbaI (6001) cPPT/CTS NheI (537) BmtI (541) ATG KflI (4043) BsaBI * (4164) FseI (4798) BamHI (4821) SpeI (5083) AscI (5172) HincII (5310) PflFI - Tth111I (5389) BsiWI (5403) RsrII (5463) BstEII (5481) SexAI * (5914) pCW-Cas9 11,885 bp
PaeR7I  (11)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (11)
1 site
C T C G A G G A G C T C
AleI  (11,525)
1 site
C A C N N N N G T G G T G N N N N C A C
BbvCI  (11,372)
1 site
C C T C A G C G G A G T C G
NotI  (11,096)
1 site
G C G G C C G C C G C C G G C G
AflIII  (9766)
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  (9766)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (9664)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
SspI  (8073)
1 site
A A T A T T T T A T A A
SfiI  (7971)
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.
KpnI  (7351)
1 site
G G T A C C C C A T G G
Acc65I  (7347)
1 site
G G T A C C C C A T G G
SmaI  (6738)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (6736)
1 site
C C C G G G G G G C C C
XmaI  (6736)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
NdeI  (6577)
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.
XbaI  (6001)
1 site
T C T A G A A G A T C T
NheI  (537)
1 site
G C T A G C C G A T C G
BmtI  (541)
1 site
G C T A G C C G A T C G
KflI  (4043)
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.
BsaBI  (4164)
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.
FseI  (4798)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
BamHI  (4821)
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.
SpeI  (5083)
1 site
A C T A G T T G A T C A
AscI  (5172)
1 site
G G C G C G C C C C G C G C G G
HincII  (5310)
1 site
G T Y R A C C A R Y T G
PflFI  (5389)
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  (5389)
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  (5403)
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  (5463)
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  (5481)
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  (5914)
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.
Cas9
669 .. 4769  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
669 .. 4769  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
AmpR
8091 .. 8951  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8091 .. 8159  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8091 .. 8951  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8160 .. 8951  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8091 .. 8951  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 1:  rTetR  
   5998 .. 6615  =  618 bp
   206 amino acids  =  23.2 kDa
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 2:  
   6616 .. 6621  =  6 bp
   2 amino acids  =  172.2 Da
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 3:  VP16 minimal transcriptional activation domain  
   6622 .. 6657  =  36 bp
   12 amino acids  =  1.4 kDa
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 4:  
   6658 .. 6660  =  3 bp
   1 amino acid  =  115.1 Da
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 5:  VP16 minimal transcriptional activation domain  
   6661 .. 6696  =  36 bp
   12 amino acids  =  1.4 kDa
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 6:  
   6697 .. 6699  =  3 bp
   1 amino acid  =  115.1 Da
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 7:  VP16 minimal transcriptional activation domain  
   6700 .. 6735  =  36 bp
   12 amino acids  =  1.4 kDa
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 8:  
   6736 .. 6744  =  9 bp
   2 amino acids  =  172.2 Da
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
rtTA-Advanced
5998 .. 6744  =  747 bp
248 amino acids  =  27.8 kDa
8 segments
Product: improved tetracycline-controlled
transactivator
In the Tet-On® system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription
only in the presence of tetracycline or doxycycline.
PuroR
5347 .. 5943  =  597 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
5347 .. 5943  =  597 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
9122 .. 9710  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9122 .. 9710  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
hPGK promoter
4827 .. 5337  =  511 bp
human phosphoglycerate kinase 1 promoter
hPGK promoter
4827 .. 5337  =  511 bp
human phosphoglycerate kinase 1 promoter
tight TRE promoter
212 .. 526  =  315 bp
Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter
tight TRE promoter
212 .. 526  =  315 bp
Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter
3' LTR (ΔU3)
7418 .. 7651  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
7418 .. 7651  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RRE
11,251 .. 11,484  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
11,251 .. 11,484  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RSV promoter
10,179 .. 10,405  =  227 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
10,179 .. 10,405  =  227 bp
Rous sarcoma virus enhancer/promoter
5' LTR (truncated)
10,406 .. 10,586  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
10,406 .. 10,586  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
SV40 ori
7884 .. 8019  =  136 bp
SV40 origin of replication
SV40 ori
7884 .. 8019  =  136 bp
SV40 origin of replication
HIV-1 Ψ
10,633 .. 10,758  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
10,633 .. 10,758  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
7723 .. 7844  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
7723 .. 7844  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
41 .. 158  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
41 .. 158  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
8021 .. 8090  =  70 bp
AmpR promoter
8021 .. 8090  =  70 bp
3xFLAG
552 .. 617  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
552 .. 617  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
T2A
5944 .. 5997  =  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
5944 .. 5997  =  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.
nucleoplasmin NLS
4770 .. 4817  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
4770 .. 4817  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
SV40 NLS
624 .. 644  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
624 .. 644  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
549 .. 551  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
549 .. 551  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
tet operator
220 .. 238  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
220 .. 238  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
256 .. 274  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
256 .. 274  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
291 .. 309  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
291 .. 309  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
327 .. 345  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
327 .. 345  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
363 .. 381  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
363 .. 381  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
398 .. 416  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
398 .. 416  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
434 .. 452  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
434 .. 452  =  19 bp
bacterial operator O2 for the tetR and tetA genes
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