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pCLIP-Cas9-Nuclease-hCMV-ZsGreen

Lentiviral CRISPR vector encoding Cas9 and the green fluorescent protein ZsGreen expressed from the human CMV promoter.

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pCLIP-Cas9-Nuclease-hCMV-ZsGreen Sequence and MappCLIP-Cas9-Nuclease-hCMV-ZsGreen.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SspI (11,040) PvuI (10,606) FspI (10,458) DrdI (9454) AflIII - PciI (9346) SmaI (9326) TspMI - XmaI (9324) AvrII (9303) SexAI * (9070) PmeI (8978) PsiI (8928) KpnI (8593) Acc65I (8589) Bsu36I (8585) SacII (8370) BsrGI (7697) PflFI - Tth111I (7381) SgrAI (7234) FLAG BsaBI * (6403) PmlI (5694) NheI (405) BmtI (409) BssHII (745) HIV-1 Ψ NruI * (867) BspQI - SapI (1136) MfeI (1219) BbvCI (1454) AleI (1607) cPPT/CTS BsmBI (2766) PspXI (2882) XbaI (2888) AfeI (2895) PspOMI (4260) ApaI (4264) PasI (4674) DraIII (5304) pCLIP-Cas9-Nuclease-hCMV-ZsGreen 11,158 bp
SspI  (11,040)
1 site
A A T A T T T T A T A A
PvuI  (10,606)
1 site
C G A T C G G C T A G C
FspI  (10,458)
1 site
T G C G C A A C G C G T
DrdI  (9454)
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.
AflIII  (9346)
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  (9346)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SmaI  (9326)
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  (9324)
1 site
C C C G G G G G G C C C
XmaI  (9324)
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.
AvrII  (9303)
1 site
C C T A G G G G A T C C
SexAI  (9070)
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.
PmeI  (8978)
1 site
G T T T A A A C C A A A T T T G
PsiI  (8928)
1 site
T T A T A A A A T A T T
KpnI  (8593)
1 site
G G T A C C C C A T G G
Acc65I  (8589)
1 site
G G T A C C C C A T G G
Bsu36I  (8585)
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.
SacII  (8370)
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.
BsrGI  (7697)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
PflFI  (7381)
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  (7381)
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.
SgrAI  (7234)
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.
BsaBI  (6403)
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.
PmlI  (5694)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NheI  (405)
1 site
G C T A G C C G A T C G
BmtI  (409)
1 site
G C T A G C C G A T C G
BssHII  (745)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
NruI  (867)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BspQI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
MfeI  (1219)
1 site
C A A T T G G T T A A C
BbvCI  (1454)
1 site
C C T C A G C G G A G T C G
AleI  (1607)
1 site
C A C N N N N G T G G T G N N N N C A C
BsmBI  (2766)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PspXI  (2882)
1 site
V C T C G A G B B G A G C T C V
XbaI  (2888)
1 site
T C T A G A A G A T C T
AfeI  (2895)
1 site
A G C G C T T C G C G A
PspOMI  (4260)
1 site
G G G C C C C C C G G G
ApaI  (4264)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PasI  (4674)
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.
DraIII  (5304)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
Cas9
2905 .. 7008  =  4104 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
2905 .. 7008  =  4104 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
10,163 .. 11,023  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,163 .. 10,954  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,163 .. 11,023  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,955 .. 11,023  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,163 .. 11,023  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ZsGreen1
7147 .. 7842  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
7147 .. 7842  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
WPRE
7867 .. 8455  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7867 .. 8455  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
9407 .. 9992  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9407 .. 9992  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
8989 .. 9318  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
8989 .. 9318  =  330 bp
SV40 enhancer and early promoter
CMV enhancer
2238 .. 2541  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2238 .. 2541  =  304 bp
human cytomegalovirus immediate early enhancer
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
RRE
1333 .. 1566  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1333 .. 1566  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
8660 .. 8893  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
8660 .. 8893  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
CMV promoter
2542 .. 2745  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
2542 .. 2745  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
2057 .. 2174  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2057 .. 2174  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
11,024 .. 11,128  =  105 bp
AmpR promoter
11,024 .. 11,128  =  105 bp
P2A
7090 .. 7146  =  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
7090 .. 7146  =  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
7009 .. 7056  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
7009 .. 7056  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
FLAG
7057 .. 7080  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
7057 .. 7080  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
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