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Plasmid Files

pICH41308::hCas9

Level 0 hCas9 module plasmid encoding human codon-optimized Cas9 with a nuclear localization signal.

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pICH41308_hCas9 Sequence and MappICH41308_hCas9.dna
Map and Sequence File   
Sequence Author:  Kamoun Lab / Addgene #49770
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 ZraI (81) AscI (6188) EcoRI (6055) PaeR7I - XhoI (5776) AarI (5493) NruI (5473) XmnI (5365) FspAI (5183) BstXI - PmlI (5022) BmgBI (4743) BamHI (4238) SacI (3803) Eco53kI (3801) SmaI (3601) TspMI - XmaI (3599) Bsu36I (3499) AatII (83) BstEII (592) BglI (1064) SnaBI (1189) AseI (2212) EcoRV (2262) NmeAIII (2338) BsmBI (2395) SbfI (2480) XcmI (2584) NdeI (2711) SalI (2773) pICH41308::hCas9 6388 bp
ZraI  (81)
1 site
G A C G T C C T G C A G
AscI  (6188)
1 site
G G C G C G C C C C G C G C G G
EcoRI  (6055)
1 site
G A A T T C C T T A A G
PaeR7I  (5776)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (5776)
1 site
C T C G A G G A G C T C
AarI  (5493)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
NruI  (5473)
1 site
T C G C G A A G C G C T
XmnI  (5365)
1 site
G A A N N N N T T C C T T N N N N A A G
FspAI  (5183)
1 site
R T G C G C A Y Y A C G C G T R
BstXI  (5022)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
PmlI  (5022)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (4743)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BamHI  (4238)
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.
SacI  (3803)
1 site
G A G C T C C T C G A G
Eco53kI  (3801)
1 site
G A G C T C C T C G A G
SmaI  (3601)
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  (3599)
1 site
C C C G G G G G G C C C
XmaI  (3599)
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.
Bsu36I  (3499)
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.
AatII  (83)
1 site
G A C G T C C T G C A G
BstEII  (592)
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.
BglI  (1064)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
SnaBI  (1189)
1 site
T A C G T A A T G C A T
AseI  (2212)
1 site
A T T A A T T A A T T A
EcoRV  (2262)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
NmeAIII  (2338)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BsmBI  (2395)
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.
SbfI  (2480)
1 site
C C T G C A G G G G A C G T C C
XcmI  (2584)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
NdeI  (2711)
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.
SalI  (2773)
1 site
G T C G A C C A G C T G
Cas9
2233 .. 6336  =  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
2233 .. 6336  =  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
SmR
434 .. 1225  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
434 .. 1225  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
ori
1396 .. 1984  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1396 .. 1984  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
110 .. 214  =  105 bp
AmpR promoter
110 .. 214  =  105 bp
SV40 NLS
6349 .. 6369  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6349 .. 6369  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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