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Plasmid Files

LSL-Cas9-Rosa26TV

Zhang lab vector with positive and negative selection markers, for targeting Cre-dependent Cas9 and EGFP expression to the mouse Rosa26 locus.

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LSL-Cas9-Rosa26TV Sequence and MapLSL-Cas9-Rosa26TV.dna
Map and Sequence File   
Sequence Author:  Zhang Lab / Addgene #61408
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 MauBI (446) KpnI (17,735) Acc65I (17,731) PaeR7I - PspXI - XhoI (17,716) BbvCI (17,090) ATG SgrDI (16,115) AvrII (14,690) AscI (11,754) NeoR/KanR PflFI - Tth111I (10,678) Rosa26 left arm PacI (1092) SnaBI (1460) chicken β- actin promoter chimeric intron BsiWI (2294) AarI (2332) MreI - SgrAI (2420) loxP loxP BstBI (3867) ATG 3xFLAG SV40 NLS KflI (7377) nucleoplasmin NLS MluI (8935) NsiI (8945) attB LSL-Cas9-Rosa26TV 20,597 bp
MauBI  (446)
1 site
C G C G C G C G G C G C G C G C
KpnI  (17,735)
1 site
G G T A C C C C A T G G
Acc65I  (17,731)
1 site
G G T A C C C C A T G G
PaeR7I  (17,716)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (17,716)
1 site
V C T C G A G B B G A G C T C V
XhoI  (17,716)
1 site
C T C G A G G A G C T C
BbvCI  (17,090)
1 site
C C T C A G C G G A G T C G
SgrDI  (16,115)
1 site
C G T C G A C G G C A G C T G C
AvrII  (14,690)
1 site
C C T A G G G G A T C C
AscI  (11,754)
1 site
G G C G C G C C C C G C G C G G
PflFI  (10,678)
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  (10,678)
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.
PacI  (1092)
1 site
T T A A T T A A A A T T A A T T
SnaBI  (1460)
1 site
T A C G T A A T G C A T
BsiWI  (2294)
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.
AarI  (2332)
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.
MreI  (2420)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (2420)
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.
BstBI  (3867)
1 site
T T C G A A A A G C T T
KflI  (7377)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
MluI  (8935)
1 site
A C G C G T T G C G C A
NsiI  (8945)
1 site
A T G C A T T A C G T A
Rosa26 right arm
11,763 .. 16,103  =  4341 bp
right homology arm from intron 1 of the mouse
Rosa26 gene (Soriano, 1999)
Rosa26 right arm
11,763 .. 16,103  =  4341 bp
right homology arm from intron 1 of the mouse
Rosa26 gene (Soriano, 1999)
Cas9
4003 .. 8103  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
4003 .. 8103  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Rosa26 left arm
3 .. 1085  =  1083 bp
left homology arm from intron 1 of the mouse
Rosa26 gene (Soriano, 1999)
Rosa26 left arm
3 .. 1085  =  1083 bp
left homology arm from intron 1 of the mouse
Rosa26 gene (Soriano, 1999)
chimeric intron
1765 .. 2782  =  1018 bp
chimera between introns from chicken β-actin and
rabbit β-globin
chimeric intron
1765 .. 2782  =  1018 bp
chimera between introns from chicken β-actin and
rabbit β-globin
AmpR
18,949 .. 19,809  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   18,949 .. 19,740  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
18,949 .. 19,809  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   19,741 .. 19,809  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
18,949 .. 19,809  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
10,423 .. 11,226  =  804 bp
267 amino acids  =  29.2 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
10,423 .. 11,226  =  804 bp
267 amino acids  =  29.2 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
EGFP
8218 .. 8934  =  717 bp
238 amino acids  =  26.8 kDa
   Segment 1:  1a  
   8218 .. 8220  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
8218 .. 8934  =  717 bp
238 amino acids  =  26.8 kDa
   Segment 2:  
   8221 .. 8934  =  714 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
8218 .. 8934  =  717 bp
238 amino acids  =  26.8 kDa
2 segments
Product: enhanced GFP
mammalian codon-optimized
WPRE
8985 .. 9573  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
8985 .. 9573  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
18,190 .. 18,778  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
18,190 .. 18,778  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
DTA
16,678 .. 17,247  =  570 bp
190 amino acids  =  21.0 kDa
Product: diphteria toxin Fragment A
DTA
16,678 .. 17,247  =  570 bp
190 amino acids  =  21.0 kDa
Product: diphteria toxin Fragment A
PGK promoter
9864 .. 10,363  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
9864 .. 10,363  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
16,152 .. 16,651  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
16,152 .. 16,651  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK poly(A) signal
11,236 .. 11,697  =  462 bp
mouse phosphoglycerate kinase 1 polyadenylation
signal
PGK poly(A) signal
11,236 .. 11,697  =  462 bp
mouse phosphoglycerate kinase 1 polyadenylation
signal
f1 ori
19,940 .. 20,395  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
19,940 .. 20,395  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
1181 .. 1484  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
1181 .. 1484  =  304 bp
human cytomegalovirus immediate early enhancer
chicken β-actin promoter
1486 .. 1763  =  278 bp
chicken β-actin promoter
1486 .. 1763  =  278 bp
bGH poly(A) signal
17,463 .. 17,687  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
17,463 .. 17,687  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
9594 .. 9801  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
9594 .. 9801  =  208 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
3115 .. 3249  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3115 .. 3249  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3369 .. 3503  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3369 .. 3503  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3623 .. 3757  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3623 .. 3757  =  135 bp
SV40 polyadenylation signal
AmpR promoter
19,810 .. 19,914  =  105 bp
AmpR promoter
19,810 .. 19,914  =  105 bp
3xFLAG
3886 .. 3951  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
3xFLAG
3886 .. 3951  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags,
followed by an enterokinase cleavage site
small t intron
17,324 .. 17,389  =  66 bp
simian virus 40 (SV40) small t antigen intron
small t intron
17,324 .. 17,389  =  66 bp
simian virus 40 (SV40) small t antigen intron
P2A
8161 .. 8217  =  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
8161 .. 8217  =  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
8104 .. 8151  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
nucleoplasmin NLS
8104 .. 8151  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from
nucleoplasmin
loxP
2915 .. 2948  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
loxP
2915 .. 2948  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
loxP
3786 .. 3819  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
loxP
3786 .. 3819  =  34 bp
Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT).
attB
9809 .. 9842  =  34 bp
minimal attB site for the φC31 integrase (Groth et
al., 2000)
attB
9809 .. 9842  =  34 bp
minimal attB site for the φC31 integrase (Groth et
al., 2000)
SV40 NLS
3958 .. 3978  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
3958 .. 3978  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
3883 .. 3885  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3883 .. 3885  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
16,669 .. 16,671  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
16,669 .. 16,671  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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