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pCLIP-Cas9-Nuclease-EFS-Puro

Lentiviral CRISPR vector encoding Cas9 and a puromycin resistance marker expressed from the EF-1α core promoter.

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pCLIP-Cas9-Nuclease-EFS-Puro Sequence and MappCLIP-Cas9-Nuclease-EFS-Puro.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SspI (10,575) PvuI (10,141) FspI (9993) DrdI (8989) AflIII - PciI (8881) SmaI (8861) TspMI - XmaI (8859) AvrII (8838) PmeI (8513) PsiI (8463) KpnI (8128) Acc65I (8124) Bsu36I (8120) PaeR7I - XhoI (8006) SalI (8000) BspDI - ClaI (7992) EcoRI (7378) BstEII (6912) RsrII (6894) BsiWI (6834) PflFI - Tth111I (6820) BamHI (6715) FLAG BsaBI * (6037) SpeI (41) CMV enhancer NdeI (276) SnaBI (382) HIV-1 Ψ NruI * (867) BspQI - SapI (1136) NotI (1178) BbvCI (1454) AleI (1607) AgeI (2516) XbaI (2522) AfeI (2529) PspOMI (3894) ApaI (3898) PmlI (5328) pCLIP-Cas9-Nuclease-EFS-Puro 10,693 bp
SspI  (10,575)
1 site
A A T A T T T T A T A A
PvuI  (10,141)
1 site
C G A T C G G C T A G C
FspI  (9993)
1 site
T G C G C A A C G C G T
DrdI  (8989)
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  (8881)
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  (8881)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SmaI  (8861)
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  (8859)
1 site
C C C G G G G G G C C C
XmaI  (8859)
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  (8838)
1 site
C C T A G G G G A T C C
PmeI  (8513)
1 site
G T T T A A A C C A A A T T T G
PsiI  (8463)
1 site
T T A T A A A A T A T T
KpnI  (8128)
1 site
G G T A C C C C A T G G
Acc65I  (8124)
1 site
G G T A C C C C A T G G
Bsu36I  (8120)
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  (8006)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (8006)
1 site
C T C G A G G A G C T C
SalI  (8000)
1 site
G T C G A C C A G C T G
BspDI  (7992)
1 site
A T C G A T T A G C T A
ClaI  (7992)
1 site
A T C G A T T A G C T A
EcoRI  (7378)
1 site
G A A T T C C T T A A G
BstEII  (6912)
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.
RsrII  (6894)
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.
BsiWI  (6834)
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.
PflFI  (6820)
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  (6820)
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.
BamHI  (6715)
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  (6037)
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
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.
NotI  (1178)
1 site
G C G G C C G C C G C C G G C G
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
AgeI  (2516)
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  (2522)
1 site
T C T A G A A G A T C T
AfeI  (2529)
1 site
A G C G C T T C G C G A
PspOMI  (3894)
1 site
G G G C C C C C C G G G
ApaI  (3898)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PmlI  (5328)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
2539 .. 6642  =  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
2539 .. 6642  =  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
9698 .. 10,558  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9698 .. 10,489  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9698 .. 10,558  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,490 .. 10,558  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9698 .. 10,558  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
PuroR
6781 .. 7377  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
6781 .. 7377  =  597 bp
198 amino acids  =  21.4 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
7402 .. 7990  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7402 .. 7990  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
8942 .. 9527  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8942 .. 9527  =  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
8524 .. 8853  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
8524 .. 8853  =  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
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)
8195 .. 8428  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
8195 .. 8428  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
EF-1α core promoter
2303 .. 2514  =  212 bp
core promoter for human elongation factor EF-1α
EF-1α core promoter
2303 .. 2514  =  212 bp
core promoter for human elongation factor EF-1α
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
2093 .. 2210  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2093 .. 2210  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
10,559 .. 10,663  =  105 bp
AmpR promoter
10,559 .. 10,663  =  105 bp
P2A
6724 .. 6780  =  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
6724 .. 6780  =  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
6643 .. 6690  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
6643 .. 6690  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
FLAG
6691 .. 6714  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
6691 .. 6714  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
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