Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012326 | LSL-Cas9-Rosa26TV | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
Cre-dependent Cas9 expression plasmid and targeting vector for the mouse Rosa26 locus. Cas9 expression is Cre-dependent and the targeting vector has positive (Neo) and negative (DTA) selection. Please note that this plasmid is prone to recombination. We recommend screening 3-5 colonies to ensure the full plasmid is intact.
- Vector Name:
- LSL-Cas9-Rosa26TV
- Antibiotic Resistance:
- Ampicillin
- Length:
- 20597 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE,
- Selection Marker:
- Neomycin/G418(Geneticin)
- Promoter:
- CAG
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 30℃
LSL-Cas9-Rosa26TV vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
LSL-Cas9-Rosa26TV vector Sequence
LOCUS LSL-Cas9-Rosa26T 20597 bp DNA circular SYN 01-JAN-1980
DEFINITION Zhang lab vector with positive and negative selection markers, for
targeting Cre-dependent Cas9 and EGFP expression to the mouse Rosa26
locus.
ACCESSION .
VERSION .
KEYWORDS LSL-Cas9-Rosa26TV
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 20597)
AUTHORS Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE,
Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S,
Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A,
Feng G, Sharp PA, Zhang F.
TITLE CRISPR-Cas9 knockin mice for genome editing and cancer modeling.
JOURNAL Cell 2014;159:440-55.
PUBMED 25263330
REFERENCE 2 (bases 1 to 20597)
AUTHORS Zhang Lab / Addgene #61408
TITLE Direct Submission
REFERENCE 3 (bases 1 to 20597)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell";
date: "2014"; volume: "159"; pages: "440-55"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT A number of sequence corrections were made based on Addgene
sequencing results and comparisons with GenBank data.
FEATURES Location/Qualifiers
source 1..20597
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 3..1085
/label=Rosa26 left arm
/note="left homology arm from intron 1 of the mouse Rosa26
gene (Soriano, 1999)"
enhancer 1181..1484
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 1486..1763
/note="chicken beta-actin promoter"
intron 1765..2782
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
protein_bind 2858..2891
/label=FRT (minimal)
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
protein_bind 2915..2948
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
polyA_signal 3115..3249
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
polyA_signal 3369..3503
/label=SV40 poly(A) signal
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
polyA_signal 3623..3757
/label=SV40 poly(A) signal
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
protein_bind 3786..3819
/label=Cre recombinase binding site
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 3883..3885
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 3886..3951
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3958..3978
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 4003..8103
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 8104..8151
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 8161..8217
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=2A peptide from porcine teschovirus-1
polyprotein
/note="P2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 8218..8931
/label=EGFP
/note="enhanced GFP"
misc_feature 8985..9573
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
polyA_signal 9594..9801
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 9809..9842
/label=attB
/note="minimal attB site for the phi-C31 integrase (Groth
et
al., 2000)"
promoter 9864..10363
/note="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
protein_bind 10383..10416
/label=FRT (minimal)
/note="supports FLP-mediated excision but not integration
(Turan and Bode, 2011)"
CDS 10423..11223
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase from Tn5"
polyA_signal 11236..11697
/label=PGK poly(A) signal
/note="mouse phosphoglycerate kinase 1 polyadenylation
signal"
misc_feature 11763..16103
/label=Rosa26 right arm
/note="right homology arm from intron 1 of the mouse Rosa26
gene (Soriano, 1999)"
promoter 16152..16651
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 16669..16671
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 16678..17247
/label=DTA
/note="diphteria toxin Fragment A"
intron 17324..17389
/label=small t intron
/note="SV40 (simian virus 40) small t antigen intron"
polyA_signal 17463..17687
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter complement(17749..17767)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter complement(17836..17866)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(17881..17902)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(18190..18778)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(18952..19809)
/label=AmpR
/note="beta-lactamase"
promoter complement(19810..19914)
/label=AmpR promoter
rep_origin complement(19940..20395)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 20537..20553
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 20563..20581
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"