Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | LSL-Cas9-Rosa26TV | Antibiotic Resistance | Ampicillin |
| Length | 20597 bp | Type | CRISPR Plasmids |
| Source | Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, | Selection Marker | Neomycin/G418(Geneticin) |
| Copy Number | High copy number | ||
LSL-Cas9-Rosa26TV vector Vector Map
Plasmid Resuspension 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 ℃.
LSL-Cas9-Rosa26TV vector Sequence
LOCUS Exported 20597 bp ds-DNA circular SYN 06-1-2016
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
COMMENT A number of sequence corrections were made based on Addgene
sequencing results and comparisons with GenBank data.
FEATURES Location/Qualifiers
source 1..20597
/organism="synthetic DNA construct"
/lab_host="Mus musculus"
/mol_type="other DNA"
misc_feature 3..1085
/note="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
/note="chimeric intron"
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
protein_bind 2915..2948
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
polyA_signal 3115..3249
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
polyA_signal 3369..3503
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
polyA_signal 3623..3757
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
protein_bind 3786..3819
/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"
/note="ATG"
/translation="M"
CDS 3886..3951
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3958..3978
/codon_start=1
/locus_tag="
"
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/note="
"
/protein_id="
"
/translation="PKKKRKV"
CDS 4003..8103
/codon_start=1
/locus_tag="
"
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/note="Cas9"
/note="generates RNA-guided double strand breaks in DNA"
/protein_id="
"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8104..8151
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 8161..8217
/codon_start=1
/product="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..8934
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
misc_feature 8985..9573
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
polyA_signal 9594..9801
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
protein_bind 9809..9842
/bound_moiety="phage phi-C31 integrase"
/note="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"
CDS 10423..11226
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 11236..11697
/note="PGK poly(A) signal"
/note="mouse phosphoglycerate kinase 1 polyadenylation
signal"
misc_feature 11763..16103
/note="Rosa26 right arm"
/note="right homology arm from intron 1 of the mouse Rosa26
gene (Soriano, 1999)"
promoter 16152..16651
/note="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 16669..16671
/codon_start=1
/product="start codon"
/note="ATG"
/translation="M"
CDS 16678..17247
/codon_start=1
/gene="Corynebacterium diphtheriae tox"
/product="diphteria toxin Fragment A"
/note="DTA"
/translation="DDVVILLIFCMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDD
DWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELG
LSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEIN
FETRGKRGQDAMYEYMAQACAGNRVRR"
intron 17324..17389
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
polyA_signal 17463..17687
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 18190..18778
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(18949..19809)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(19810..19914)
/gene="bla"
/note="AmpR promoter"
rep_origin 19940..20395
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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