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Plasmid Files

pHAGE TRE dCas9

Lentiviral CRISPR activation vector for doxycycline-inducible expression of catalytically inactive dCas9.

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pHAGE TRE dCas9 Sequence and MappHAGE TRE dCas9.dna
Map and Sequence File   
Sequence Author:  Maehr & Wolfe Labs / Addgene #50915
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 SV40 NLS SgrAI (14,248) AscI (14,096) FspAI (13,091) Bsu36I (11,407) BlpI (11,089) SalI (10,681) SbfI (10,388) attB1 minimal CMV promoter AleI (8983) BstAPI (8859) BbvCI (8830) FseI (8556) BstZ17I (14,277) PspXI (39) NdeI (2028) SphI (3356) RsrII (3467) AhdI (5789) SfiI (6909) pHAGE TRE dCas9 14,427 bp
SgrAI  (14,248)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
AscI  (14,096)
1 site
G G C G C G C C C C G C G C G G
FspAI  (13,091)
1 site
R T G C G C A Y Y A C G C G T R
Bsu36I  (11,407)
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.
BlpI  (11,089)
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.
SalI  (10,681)
1 site
G T C G A C C A G C T G
SbfI  (10,388)
1 site
C C T G C A G G G G A C G T C C
AleI  (8983)
1 site
C A C N N N N G T G G T G N N N N C A C
BstAPI  (8859)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
BbvCI  (8830)
1 site
C C T C A G C G G A G T C G
FseI  (8556)
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.
BstZ17I  (14,277)
1 site
G T A T A C C A T A T G
PspXI  (39)
1 site
V C T C G A G B B G A G C T C V
NdeI  (2028)
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.
SphI  (3356)
1 site
G C A T G C C G T A C G
RsrII  (3467)
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.
AhdI  (5789)
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.
SfiI  (6909)
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.
dCas9
10,141 .. 14,244  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   10,141 .. 10,167  =  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
10,141 .. 14,244  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   10,168 .. 10,170  =  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
10,141 .. 14,244  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   10,171 .. 12,657  =  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
10,141 .. 14,244  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   12,658 .. 12,660  =  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
10,141 .. 14,244  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   12,661 .. 14,244  =  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
10,141 .. 14,244  =  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
UbC promoter
179 .. 1390  =  1212 bp
human ubiquitin C promoter
UbC promoter
179 .. 1390  =  1212 bp
human ubiquitin C promoter
AmpR
5002 .. 5862  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5002 .. 5070  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5002 .. 5862  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   5071 .. 5862  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5002 .. 5862  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
2823 .. 3617  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
2823 .. 3617  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
rtTA-Advanced
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 1:  rTetR  
   1449 .. 2066  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 2:  
   2067 .. 2072  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 3:  VP16 minimal transcriptional activation domain  
   2073 .. 2108  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 4:  
   2109 .. 2111  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 5:  VP16 minimal transcriptional activation domain  
   2112 .. 2147  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 6:  
   2148 .. 2150  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 7:  VP16 minimal transcriptional activation domain  
   2151 .. 2186  =  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
1449 .. 2195  =  747 bp
248 amino acids  =  27.8 kDa
   Segment 8:  
   2187 .. 2195  =  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
1449 .. 2195  =  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.
3' LTR
7407 .. 8040  =  634 bp
3' long terminal repeat (LTR) from HIV-1
3' LTR
7407 .. 8040  =  634 bp
3' long terminal repeat (LTR) from HIV-1
WPRE
3796 .. 4384  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
3796 .. 4384  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
6033 .. 6621  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6033 .. 6621  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
2233 .. 2807  =  575 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
2233 .. 2807  =  575 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 1:  tetO  
   9636 .. 9654  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 2:  
   9655 .. 9677  =  23 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 3:  tetO  
   9678 .. 9696  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 4:  
   9697 .. 9719  =  23 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 5:  tetO  
   9720 .. 9738  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 6:  
   9739 .. 9761  =  23 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 7:  tetO  
   9762 .. 9780  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 8:  
   9781 .. 9803  =  23 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 9:  tetO  
   9804 .. 9822  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 10:  
   9823 .. 9845  =  23 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 11:  tetO  
   9846 .. 9864  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 12:  
   9865 .. 9887  =  23 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
   Segment 13:  tetO  
   9888 .. 9906  =  19 bp
contains seven copies of the tetracycline operator
tetO
tetracycline response element
9636 .. 9906  =  271 bp
13 segments
contains seven copies of the tetracycline operator
tetO
3' LTR (ΔU3)
4459 .. 4692  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
4459 .. 4692  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
RRE
8709 .. 8942  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
8709 .. 8942  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
HIV-1 Ψ
8087 .. 8212  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
8087 .. 8212  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
9464 .. 9581  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
9464 .. 9581  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
4897 .. 5001  =  105 bp
AmpR promoter
4897 .. 5001  =  105 bp
3xHA
14,278 .. 14,367  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
3xHA
14,278 .. 14,367  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
minimal CMV promoter
9939 .. 9977  =  39 bp
human cytomegalovirus (CMV) immediate early
promoter
minimal CMV promoter
9939 .. 9977  =  39 bp
human cytomegalovirus (CMV) immediate early
promoter
attB1
10,107 .. 10,131  =  25 bp
recombination site for the Gateway® BP reaction
attB1
10,107 .. 10,131  =  25 bp
recombination site for the Gateway® BP reaction
attB2
14,417 .. 14  =  25 bp
recombination site for the Gateway® BP reaction
attB2
14,417 .. 14  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
14,257 .. 14,277  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
14,257 .. 14,277  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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