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lentiCas9n(D10A)-Blast

Zhang lab lentiviral vector with a blasticidin resistance marker, for expressing Cas9 with the D10A nickase mutation.

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lentiCas9n(D10A)-Blast Sequence and MaplentiCas9n(D10A)-Blast.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #63593
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 AmpR promoter SspI (12,744) FspI (12,162) PciI (11,047) BstZ17I (10,668) FseI (10,308) SgrAI (10,146) MauBI (10,068) AvrII (9903) StuI (9902) PmeI (8855) 5' LTR (truncated) KpnI (8278) Acc65I (8274) Bsu36I (8270) PaeR7I - XhoI (8156) SacII (8055) EcoRI (7528) BamHI (7063) SgrDI (1) MluI (231) SpeI (252) NdeI (487) SnaBI (593) KasI (1018) NarI (1019) SfoI (1020) PluTI (1022) NotI (1525) BbvCI (1801) AleI (1954) PacI (2598) NheI (2602) BmtI (2606) AgeI (2864) XbaI (2870) AfeI (2877) PasI (4656) FLAG lentiCas9n(D10A)-Blast 12,860 bp
SspI  (12,744)
1 site
A A T A T T T T A T A A
FspI  (12,162)
1 site
T G C G C A A C G C G T
PciI  (11,047)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BstZ17I  (10,668)
1 site
G T A T A C C A T A T G
FseI  (10,308)
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.
SgrAI  (10,146)
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.
MauBI  (10,068)
1 site
C G C G C G C G G C G C G C G C
AvrII  (9903)
1 site
C C T A G G G G A T C C
StuI  (9902)
1 site
A G G C C T T C C G G A
PmeI  (8855)
1 site
G T T T A A A C C A A A T T T G
KpnI  (8278)
1 site
G G T A C C C C A T G G
Acc65I  (8274)
1 site
G G T A C C C C A T G G
Bsu36I  (8270)
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.
PaeR7I  (8156)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (8156)
1 site
C T C G A G G A G C T C
SacII  (8055)
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.
EcoRI  (7528)
1 site
G A A T T C C T T A A G
BamHI  (7063)
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.
SgrDI  (1)
1 site
C G T C G A C G G C A G C T G C
MluI  (231)
1 site
A C G C G T T G C G C A
SpeI  (252)
1 site
A C T A G T T G A T C A
NdeI  (487)
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.
SnaBI  (593)
1 site
T A C G T A A T G C A T
KasI  (1018)
1 site
G G C G C C C C G C G G
NarI  (1019)
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  (1020)
1 site
G G C G C C C C G C G G
PluTI  (1022)
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.
NotI  (1525)
1 site
G C G G C C G C C G C C G G C G
BbvCI  (1801)
1 site
C C T C A G C G G A G T C G
AleI  (1954)
1 site
C A C N N N N G T G G T G N N N N C A C
PacI  (2598)
1 site
T T A A T T A A A A T T A A T T
NheI  (2602)
1 site
G C T A G C C G A T C G
BmtI  (2606)
1 site
G C T A G C C G A T C G
AgeI  (2864)
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.
XbaI  (2870)
1 site
T C T A G A A G A T C T
AfeI  (2877)
1 site
A G C G C T T C G C G A
PasI  (4656)
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.
Cas9(D10A)
2887 .. 6990  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 1:  
   2887 .. 2913  =  27 bp
   9 amino acids  =  1.1 kDa
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
2887 .. 6990  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 2:  
   2914 .. 2916  =  3 bp
   1 amino acid  =  89.1 Da
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
2887 .. 6990  =  4104 bp
1368 amino acids  =  158.4 kDa
   Segment 3:  
   2917 .. 6990  =  4074 bp
   1358 amino acids  =  157.3 kDa
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
Cas9(D10A)
2887 .. 6990  =  4104 bp
1368 amino acids  =  158.4 kDa
3 segments
Product: nickase mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II
CRISPR/Cas system
generates RNA-guided single strand nicks in DNA
due to the D10A mutation in the RuvC catalytic
domain
AmpR
11,867 .. 12,727  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   11,867 .. 12,658  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
11,867 .. 12,727  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   12,659 .. 12,727  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
11,867 .. 12,727  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
WPRE
7552 .. 8140  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7552 .. 8140  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
11,108 .. 11,696  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
11,108 .. 11,696  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
9147 .. 9575  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
9147 .. 9575  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
7129 .. 7527  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
7129 .. 7527  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
238 .. 617  =  380 bp
human cytomegalovirus immediate early enhancer
BleoR
10,032 .. 10,406  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
10,032 .. 10,406  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
SV40 promoter
9589 .. 9918  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
9589 .. 9918  =  330 bp
SV40 enhancer and early promoter
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1680 .. 1913  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
bGH poly(A) signal
8877 .. 9101  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
8877 .. 9101  =  225 bp
bovine growth hormone polyadenylation signal
EF-1α core promoter
2651 .. 2862  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
2651 .. 2862  =  212 bp
core promoter for human elongation factor EF-1α
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
619 .. 817  =  199 bp
human cytomegalovirus (CMV) immediate early
promoter
5' LTR (truncated)
835 .. 1015  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
835 .. 1015  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
8665 .. 8845  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
8665 .. 8845  =  181 bp
truncated 5' long terminal repeat (LTR) from HIV-1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
1062 .. 1187  =  126 bp
packaging signal of human immunodeficiency virus
type 1
SV40 poly(A) signal
10,536 .. 10,657  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
10,536 .. 10,657  =  122 bp
SV40 polyadenylation signal
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2440 .. 2557  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
12,728 .. 12,832  =  105 bp
AmpR promoter
12,728 .. 12,832  =  105 bp
P2A
7072 .. 7128  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
P2A
7072 .. 7128  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
nucleoplasmin NLS
6991 .. 7038  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6991 .. 7038  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
EM7 promoter
9966 .. 10,013  =  48 bp
synthetic bacterial promoter
EM7 promoter
9966 .. 10,013  =  48 bp
synthetic bacterial promoter
FLAG
7039 .. 7062  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
7039 .. 7062  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 ori
9769 .. 9904  =  136 bp
SV40 origin of replication
SV40 ori
9769 .. 9904  =  136 bp
SV40 origin of replication
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