LOCUS       Exported                8986 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCMV-CBA-Cas9-Rosa26gRNAs, complete sequence.
ACCESSION   MG550105
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8986)
  AUTHORS   Kozhevnikova EN, Leshchenko AE, Pindyurin AV.
  TITLE     An inducible DamID system for profiling of a nuclear lamina protein 
            component Lamin B1 interactions with chromosomes in mouse cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8986)
  AUTHORS   Kozhevnikova EN, Leshchenko AE, Pindyurin AV.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-NOV-2017) Department of the Regulation of Genetic 
            Processes, Institute of Molecular and Cellular Biology, Lavrentyev 
            ave. 8/2, Novosibirsk, Novosibirsk Region 630090, Russia
REFERENCE   3  (bases 1 to 8986)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8986
                     /organism="Cloning vector pCMV-CBA-Cas9-Rosa26gRNAs"
                     /mol_type="other DNA"
                     /note="derived from Addgene plasmid #64073 (pX333) v. 
                     16-NOV-2017"
                     /db_xref="taxon:2066508"
     regulatory      70..318
                     /regulatory_class="promoter"
                     /note="human U6 promoter"
     promoter        70..310
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     ncRNA           319..420
                     /product="guide RNA #2; specific to mouse Rosa26 locus"
                     /label=guide_RNA
                     /ncRNA_class="guide_RNA"
     misc_feature    319..338
                     /label=Rosa26 gRNA2 sequence
                     /note="Rosa26 gRNA2 sequence"
     misc_feature    339..414
                     /note="guide RNA scaffold for the CRISPR/Cas9 system; gRNA 
                     scaffold"
     misc_RNA        339..414
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      415..420
                     /regulatory_class="terminator"
                     /note="RNA polymerase III transcription terminator; polIII 
                     terminator"
     regulatory      505..753
                     /regulatory_class="promoter"
                     /note="human U6 promoter"
     promoter        505..745
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     ncRNA           754..855
                     /product="guide RNA #1; specific to mouse Rosa26 locus"
                     /label=guide_RNA
                     /ncRNA_class="guide_RNA"
     misc_feature    754..773
                     /label=Rosa26 gRNA1 sequence
                     /note="Rosa26 gRNA1 sequence"
     misc_feature    774..849
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system; gRNA scaffold"
     misc_RNA        774..849
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      850..855
                     /regulatory_class="terminator"
                     /note="RNA polymerase III transcription terminator; polIII 
                     terminator"
     regulatory      939..1224
                     /regulatory_class="enhancer"
                     /note="human cytomegalovirus immediate early gene enhancer;
                     CMV enhancer"
     enhancer        939..1224
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     regulatory      1226..1503
                     /regulatory_class="promoter"
                     /note="chicken beta-actin promoter"
     promoter        1226..1503
                     /label=chicken beta-actin promoter
     intron          1504..1731
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1743..1752
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1749..6020
                     /codon_start=1
                     /product="FLAG-Cas9 nuclease"
                     /label=FLAG-Cas9 nuclease
                     /note="FLAG tagged; includes nuclear localization 
                     sequences"
                     /protein_id="AUM57420.1"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYS
                     IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK
                     RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD
                     EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK
                     LFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI
                     ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL
                     LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA
                     GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL
                     HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF
                     EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP
                     AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD
                     LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR
                     YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS
                     GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG
                     QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
                     LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL
                     ITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI
                     REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV
                     YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGE
                     TGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD
                     PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG
                     YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL
                     KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE
                     QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS
                     QLGGDKRPAATKKAGQAKKKK"
     misc_feature    1752..1817
                     /label=Region: 3xFLAG
                     /note="Region: 3xFLAG"
     CDS             1752..1817
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1824..1844
                     /label=Region: nuclear localization signal (NLS)
                     /note="Region: nuclear localization signal (NLS)"
     CDS             1824..1844
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    1869..5969
                     /label=Region: Cas9 nuclease
                     /note="Region: Cas9 nuclease"
     CDS             1869..5969
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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"
     misc_feature    5970..6017
                     /label=Region: nuclear localization signal (NLS)
                     /note="Region: nuclear localization signal (NLS)"
     CDS             5970..6017
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     regulatory      6051..6258
                     /regulatory_class="terminator"
                     /note="bovine growth hormone gene polyadenylation signal; 
                     bGH polyA"
     polyA_signal    6051..6258
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6267..6407
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6482..6937
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        7219..7323
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7324..8184
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="ampicillin resistance protein"
                     /protein_id="AUM57421.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            7324..8184
                     /gene="bla"
                     /label=bla
     rep_origin      8355..8943
                     /direction=RIGHT
                     /note="high-copy-number; ColE1/pMB1/pBR322/pUC"