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Plasmid Files

pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST

Level 1 hCas9 module plasmid for expressing human codon-optimized Cas9 from the CaMV 35S  promoter.

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pICH47742_2x35S-5pICH47742_2x35S-5'UTR-hCas9(STOP)-NOST.dna
Map and Sequence File   
Sequence Author:  Kamoun Lab / Addgene #49771
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 PspOMI (9615) SacII (9334) NotI (9328) AseI (7994) AhdI (7822) SfiI (6302) AleI (5615) SexAI * (5522) SphI (5436) PmeI (5311) MauBI (5250) NsiI (5146) SV40 NLS ApaI (9619) PspXI (786) BspDI - ClaI (866) EcoRV (903) BsmBI (1036) SbfI (1121) SalI (1414) BlpI (1822) Bsu36I (2140) TspMI - XmaI (2240) SmaI (2242) BamHI (2879) BstXI - PmlI (3663) FspAI (3824) NruI (4114) AscI (4829) pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST 9623 bp
PspOMI  (9615)
1 site
G G G C C C C C C G G G
SacII  (9334)
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.
NotI  (9328)
1 site
G C G G C C G C C G C C G G C G
AseI  (7994)
1 site
A T T A A T T A A T T A
AhdI  (7822)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
SfiI  (6302)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
AleI  (5615)
1 site
C A C N N N N G T G G T G N N N N C A C
SexAI  (5522)
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.
SphI  (5436)
1 site
G C A T G C C G T A C G
PmeI  (5311)
1 site
G T T T A A A C C A A A T T T G
MauBI  (5250)
1 site
C G C G C G C G G C G C G C G C
NsiI  (5146)
1 site
A T G C A T T A C G T A
ApaI  (9619)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspXI  (786)
1 site
V C T C G A G B B G A G C T C V
BspDI  (866)
1 site
A T C G A T T A G C T A
ClaI  (866)
1 site
A T C G A T T A G C T A
EcoRV  (903)
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.
BsmBI  (1036)
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  (1121)
1 site
C C T G C A G G G G A C G T C C
SalI  (1414)
1 site
G T C G A C C A G C T G
BlpI  (1822)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Bsu36I  (2140)
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.
TspMI  (2240)
1 site
C C C G G G G G G C C C
XmaI  (2240)
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.
SmaI  (2242)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (2879)
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.
BstXI  (3663)
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  (3663)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FspAI  (3824)
1 site
R T G C G C A Y Y A C G C G T R
NruI  (4114)
1 site
T C G C G A A G C G C T
AscI  (4829)
1 site
G G C G C G C C C C G C G C G G
Cas9
874 .. 4977  =  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
874 .. 4977  =  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
trfA
5506 .. 6654  =  1149 bp
382 amino acids  =  43.8 kDa
Product: trans-acting replication protein that binds
to and activates oriV
trfA
5506 .. 6654  =  1149 bp
382 amino acids  =  43.8 kDa
Product: trans-acting replication protein that binds
to and activates oriV
AmpR
7749 .. 8609  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7749 .. 8540  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7749 .. 8609  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   8541 .. 8609  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7749 .. 8609  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
oriV
8801 .. 9432  =  632 bp
incP origin of replication
oriV
8801 .. 9432  =  632 bp
incP origin of replication
ori
6990 .. 7578  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6990 .. 7578  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CaMV 35S promoter
438 .. 783  =  346 bp
strong constitutive promoter from cauliflower
mosaic virus
CaMV 35S promoter
438 .. 783  =  346 bp
strong constitutive promoter from cauliflower
mosaic virus
NOS terminator
5026 .. 5278  =  253 bp
nopaline synthase terminator and poly(A) signal
NOS terminator
5026 .. 5278  =  253 bp
nopaline synthase terminator and poly(A) signal
AmpR promoter
8610 .. 8714  =  105 bp
AmpR promoter
8610 .. 8714  =  105 bp
RB T-DNA repeat
5326 .. 5350  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
5326 .. 5350  =  25 bp
right border repeat from nopaline C58 T-DNA
LB T-DNA repeat
9524 .. 9548  =  25 bp
left border repeat from nopaline C58 T-DNA
LB T-DNA repeat
9524 .. 9548  =  25 bp
left border repeat from nopaline C58 T-DNA
SV40 NLS
4990 .. 5010  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4990 .. 5010  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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