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pCLIP-Cas9-Nuclease-TRE3G-ZsGreen

Lentiviral CRISPR vector encoding Cas9 and the green fluorescent protein ZsGreen expressed from a Tet-responsive promoter.

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pCLIP-Cas9-Nuclease-TRE3G-ZsGreen Sequence and MappCLIP-Cas9-Nuclease-TRE3G-ZsGreen.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SspI (10,775) PvuI (10,341) FspI (10,193) DrdI (9189) AflIII - PciI (9081) SmaI (9061) TspMI - XmaI (9059) AvrII (9038) SexAI * (8805) PmeI (8713) PsiI (8663) KpnI (8328) Acc65I (8324) Bsu36I (8320) SacII (8105) EcoRI (7578) BsrGI (7432) PflFI - Tth111I (7116) SgrAI (6969) BamHI (6816) FLAG BsaBI * (6138) SpeI (41) CMV enhancer NdeI (276) SnaBI (382) NheI (405) BmtI (409) BssHII (745) HIV-1 Ψ NruI * (867) BspQI - SapI (1136) FseI (1184) MfeI (1223) BbvCI (1458) AleI (1611) AbsI - PspXI (2238) XbaI (2623) AfeI (2630) PspOMI (3995) ApaI (3999) PasI (4409) DraIII (5039) PmlI (5429) pCLIP-Cas9-Nuclease-TRE3G-ZsGreen 10,893 bp
SspI  (10,775)
1 site
A A T A T T T T A T A A
PvuI  (10,341)
1 site
C G A T C G G C T A G C
FspI  (10,193)
1 site
T G C G C A A C G C G T
DrdI  (9189)
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  (9081)
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  (9081)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SmaI  (9061)
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  (9059)
1 site
C C C G G G G G G C C C
XmaI  (9059)
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  (9038)
1 site
C C T A G G G G A T C C
SexAI  (8805)
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  (8713)
1 site
G T T T A A A C C A A A T T T G
PsiI  (8663)
1 site
T T A T A A A A T A T T
KpnI  (8328)
1 site
G G T A C C C C A T G G
Acc65I  (8324)
1 site
G G T A C C C C A T G G
Bsu36I  (8320)
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  (8105)
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  (7578)
1 site
G A A T T C C T T A A G
BsrGI  (7432)
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  (7116)
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  (7116)
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  (6969)
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.
BamHI  (6816)
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.
BsaBI  (6138)
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.
SpeI  (41)
1 site
A C T A G T T G A T C A
NdeI  (276)
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  (382)
1 site
T A C G T A A T G C A T
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.
FseI  (1184)
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.
MfeI  (1223)
1 site
C A A T T G G T T A A C
BbvCI  (1458)
1 site
C C T C A G C G G A G T C G
AleI  (1611)
1 site
C A C N N N N G T G G T G N N N N C A C
AbsI  (2238)
1 site
C C T C G A G G G G A G C T C C
PspXI  (2238)
1 site
V C T C G A G B B G A G C T C V
XbaI  (2623)
1 site
T C T A G A A G A T C T
AfeI  (2630)
1 site
A G C G C T T C G C G A
PspOMI  (3995)
1 site
G G G C C C C C C G G G
ApaI  (3999)
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  (4409)
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  (5039)
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.
PmlI  (5429)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
2640 .. 6743  =  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
2640 .. 6743  =  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
9898 .. 10,758  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9898 .. 10,689  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9898 .. 10,758  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,690 .. 10,758  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9898 .. 10,758  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ZsGreen1
6882 .. 7577  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
6882 .. 7577  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
WPRE
7602 .. 8190  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7602 .. 8190  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
9142 .. 9727  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9142 .. 9727  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
TRE3G promoter
2244 .. 2622  =  379 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On® 3G
TRE3G promoter
2244 .. 2622  =  379 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On® 3G
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
8724 .. 9053  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
8724 .. 9053  =  330 bp
SV40 enhancer and early promoter
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
1337 .. 1570  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1337 .. 1570  =  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)
8395 .. 8628  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
8395 .. 8628  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
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
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
10,759 .. 10,863  =  105 bp
AmpR promoter
10,759 .. 10,863  =  105 bp
P2A
6825 .. 6881  =  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
6825 .. 6881  =  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
6744 .. 6791  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6744 .. 6791  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
FLAG
6792 .. 6815  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
6792 .. 6815  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
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