LOCUS       40924_11606        8986 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCMV-CBA-Cas9-Rosa26gRNAs, complete sequence.
ACCESSION   .
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  JOURNAL   Unpublished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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8986)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "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"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..310
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    319..338
                     /label=Rosa26 gRNA2 sequence
                     /note="Rosa26 gRNA2 sequence"
     misc_RNA        339..414
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      415..420
                     /note="RNA polymerase III transcription terminator; polIII 
                     terminator"
                     /regulatory_class="terminator"
     regulatory      505..753
                     /label=human U6 promoter
                     /note="human U6 promoter"
                     /regulatory_class="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
                     /note="RNA polymerase III transcription terminator; polIII 
                     terminator"
                     /regulatory_class="terminator"
     enhancer        939..1224
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     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
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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"
     CDS             1869..5969
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     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"
     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
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7219..7323
                     /label=AmpR promoter
     CDS             7324..8181
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      8355..8943
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggccttttgc tcacatgcgc cattctcgag catgcgccat tctcgagcat gctcgagaat
       61 ggcgcatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      121 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
      181 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
      241 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
      301 tggaaaggac gaaacaccaa agcaagtaat gataatttgt tttagagcta gaaatagcaa
      361 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      421 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca
      481 attctgcaga caaatggctc tagagagggc ctatttccca tgattccttc atatttgcat
      541 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      601 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      661 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      721 gctttatata tcttgtggaa aggacgaaac accaaagaca ttaaagtctg catgttttag
      781 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      841 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
      901 ttagcgcgtg cgccaattct gcagacaaat ggggtacccg ttacataact tacggtaaat
      961 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata
     1021 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1081 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1141 gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1201 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     1261 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1321 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
     1381 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     1441 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     1501 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     1561 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     1621 cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg
     1681 gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg
     1741 gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
     1801 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     1861 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     1921 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     1981 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     2041 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     2101 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     2161 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     2221 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     2281 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     2341 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     2401 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     2461 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     2521 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     2581 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     2641 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     2701 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     2761 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     2821 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     2881 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     2941 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     3001 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     3061 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     3121 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     3181 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     3241 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     3301 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     3361 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     3421 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     3481 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     3541 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     3601 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     3661 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     3721 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     3781 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     3841 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     3901 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     3961 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     4021 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     4081 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     4141 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     4201 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     4261 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     4321 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     4381 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     4441 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     4501 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     4561 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     4621 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     4681 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     4741 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     4801 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     4861 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     4921 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     4981 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     5041 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     5101 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     5161 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     5221 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     5281 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     5341 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     5401 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     5461 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     5521 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     5581 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     5641 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     5701 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     5761 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     5821 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     5881 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     5941 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     6001 aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga tcagcctcga ctgtgccttc
     6061 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     6121 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     6181 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa
     6241 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc
     6301 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     6361 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     6421 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     6481 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     6541 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     6601 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     6661 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     6721 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     6781 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     6841 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     6901 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     6961 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     7021 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     7081 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     7141 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     7201 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     7261 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     7321 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     7381 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     7441 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     7501 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     7561 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     7621 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     7681 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     7741 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     7801 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     7861 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     7921 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     7981 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     8041 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     8101 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     8161 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     8221 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     8281 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     8341 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     8401 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     8461 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     8521 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     8581 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     8641 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     8701 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     8761 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     8821 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     8881 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     8941 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgct
//
