Resources
Plasmid Files

pcDNA3.1-CibN-dCas9-CibN

Plasmid encoding two copies of CIBN fused to catalytically inactive dCas9, for use in the light-activated CRISPR-Cas9 effector (LACE) system.

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

pcDNA3.1-CibN-dCas9-CibN Sequence and MappcDNA3.1-CibN-dCas9-CibN.dna
Map and Sequence File   
Sequence Author:  Gersbach Lab / Addgene #60553
Download Free Trial Get SnapGene Viewer

 PmeI (10,320) AflII (10,312) AscI (9788) PmlI (7929) KpnI (7414) Acc65I (7410) EcoRI (6976) AleI (6883) BsgI (6259) SwaI (6157) BmgBI (5856) SacII (5633) SV40 NLS DraIII (212) SexAI * (502) StuI (734) AvrII (735) AvaI - BsoBI - TspMI - XmaI (756) BmeT110I (757) SmaI (758) EagI (851) KasI (944) NarI (945) SfoI (946) PluTI (948) PstI (998) MscI (1027) PflFI - Tth111I (1063) RsrII (1461) PfoI (1720) AhdI (3186) PvuI (3556) SgrDI (4107) BglII (4121) SpeI (4358) SnaBI (4699) ATG BspDI - ClaI (5056) pcDNA3.1-CibN-dCas9-CibN 10,632 bp
PmeI  (10,320)
1 site
G T T T A A A C C A A A T T T G
AflII  (10,312)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
AscI  (9788)
1 site
G G C G C G C C C C G C G C G G
PmlI  (7929)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
KpnI  (7414)
1 site
G G T A C C C C A T G G
Acc65I  (7410)
1 site
G G T A C C C C A T G G
EcoRI  (6976)
1 site
G A A T T C C T T A A G
AleI  (6883)
1 site
C A C N N N N G T G G T G N N N N C A C
BsgI  (6259)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
SwaI  (6157)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
BmgBI  (5856)
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.
SacII  (5633)
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.
DraIII  (212)
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.
SexAI  (502)
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.
StuI  (734)
1 site
A G G C C T T C C G G A
AvrII  (735)
1 site
C C T A G G G G A T C C
AvaI  (756)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (756)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (756)
1 site
C C C G G G G G G C C C
XmaI  (756)
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.
BmeT110I  (757)
1 site
C Y C G R G G R G C Y C
SmaI  (758)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
EagI  (851)
1 site
C G G C C G G C C G G C
KasI  (944)
1 site
G G C G C C C C G C G G
NarI  (945)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (946)
1 site
G G C G C C C C G C G G
PluTI  (948)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
PstI  (998)
1 site
C T G C A G G A C G T C
MscI  (1027)
1 site
T G G C C A A C C G G T
PflFI  (1063)
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  (1063)
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.
RsrII  (1461)
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.
PfoI  (1720)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
AhdI  (3186)
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.
PvuI  (3556)
1 site
C G A T C G G C T A G C
SgrDI  (4107)
1 site
C G T C G A C G G C A G C T G C
BglII  (4121)
1 site
A G A T C T T C T A G A
SpeI  (4358)
1 site
A C T A G T T G A T C A
SnaBI  (4699)
1 site
T A C G T A A T G C A T
BspDI  (5056)
1 site
A T C G A T T A G C T A
ClaI  (5056)
1 site
A T C G A T T A G C T A
dCas9
5637 .. 9740  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   5637 .. 5663  =  27 bp
   9 amino acids  =  1.1 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
5637 .. 9740  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   5664 .. 5666  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
5637 .. 9740  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   5667 .. 8153  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
5637 .. 9740  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   8154 .. 8156  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
5637 .. 9740  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   8157 .. 9740  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
5637 .. 9740  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
AmpR
3113 .. 3973  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3113 .. 3904  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3113 .. 3973  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3905 .. 3973  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3113 .. 3973  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
817 .. 1611  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
817 .. 1611  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
2357 .. 2942  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2357 .. 2942  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CIBN
9798 .. 10,307  =  510 bp
170 amino acids  =  19.1 kDa
Product: N-terminal portion of the CIB1 transcription
factor from Arabidopsis thaliana
binds to CRY2 (cryptochrome 2) that has absorbed
blue light (Kennedy et al., 2010)
CIBN
9798 .. 10,307  =  510 bp
170 amino acids  =  19.1 kDa
Product: N-terminal portion of the CIB1 transcription
factor from Arabidopsis thaliana
binds to CRY2 (cryptochrome 2) that has absorbed
blue light (Kennedy et al., 2010)
CIBN
5064 .. 5570  =  507 bp
169 amino acids  =  18.9 kDa
Product: N-terminal portion of the CIB1 transcription
factor from Arabidopsis thaliana
binds to CRY2 (cryptochrome 2) that has absorbed
blue light (Kennedy et al., 2010)
CIBN
5064 .. 5570  =  507 bp
169 amino acids  =  18.9 kDa
Product: N-terminal portion of the CIB1 transcription
factor from Arabidopsis thaliana
binds to CRY2 (cryptochrome 2) that has absorbed
blue light (Kennedy et al., 2010)
f1 ori
10,611 .. 407  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
10,611 .. 407  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
4344 .. 4723  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4344 .. 4723  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
421 .. 750  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
421 .. 750  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
10,341 .. 10,565  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
10,341 .. 10,565  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
4724 .. 4927  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
4724 .. 4927  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
1785 .. 1906  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1785 .. 1906  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3974 .. 4078  =  105 bp
AmpR promoter
3974 .. 4078  =  105 bp
FLAG
5577 .. 5600  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
5577 .. 5600  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
SV40 NLS
5607 .. 5627  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5607 .. 5627  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
9765 .. 9785  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
9765 .. 9785  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
5013 .. 5015  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5013 .. 5015  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
601 .. 736  =  136 bp
SV40 origin of replication
SV40 ori
601 .. 736  =  136 bp
SV40 origin of replication
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2016 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter