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"