LOCUS       Exported                7667 bp ds-DNA     circular SYN 25-OCT-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7667)
  AUTHORS   Zhenya
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Oct 25, 2019 from SnapGene 4.3.6
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7667
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     misc_feature    1..130
                     /label=AAV_ITR?
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             765..830
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             999..4253
                     /codon_start=1
                     /product="http://www.uniprot.org/uniprot/J7RUA5"
                     /label=S.a. Cas9 ORF
                     /translation="PKKKRKVGIHGVPAAKRNYILGLAIGITSVGYGIIDYETRDVIDA
                     GVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYE
                     ARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKY
                     VAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETR
                     RTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVI
                     TRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNL
                     KVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN
                     LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDF
                     ILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNE
                     RIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRS
                     VSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKT
                     KKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF
                     TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAE
                     SMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGN
                     TLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKY
                     YEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDV
                     YLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKIN
                     GELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDI
                     LGNLYEVKSKKHPQIIKKGKRPAATKKAGQAKKKKGS"
     CDS             999..1019
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4200..4247
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4254..4403
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     polyA_signal    4432..4553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_site      4438..4564
                     /label=SV40 Poly A Site
     promoter        4572..4812
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     repeat_region   4930..5070
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5145..5600
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5882..5986
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5987..6847
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7018..7606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggactac aaagaccatg
      781 acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag cacggtccgc
      841 gggctgacgc attggacgat tttgatctgg atatgctggg aagtgacgcc ctcgatgatt
      901 ttgaccttga catgcttggt tcggatgccc ttgatgactt tgacctcgac atgctcggca
      961 gtgacgccct tgatgatttc gacctggaca tggttaaccc aaagaagaag cggaaggtcg
     1021 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggcc atcggcatca
     1081 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc
     1141 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca
     1201 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact
     1261 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga
     1321 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca
     1381 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca
     1441 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc
     1501 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca
     1561 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg
     1621 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg
     1681 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga
     1741 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg
     1801 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga
     1861 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc
     1921 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca
     1981 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg
     2041 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga
     2101 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg
     2161 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca
     2221 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc
     2281 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga
     2341 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct
     2401 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca
     2461 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga
     2521 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca
     2581 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt
     2641 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg
     2701 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc
     2761 tcgtgaagca ggaagaagcc agcaagaagg gcaaccggac cccattccag tacctgagca
     2821 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg
     2881 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca
     2941 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca
     3001 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga
     3061 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc
     3121 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca
     3181 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg
     3241 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct
     3301 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc
     3361 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg
     3421 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg
     3481 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc
     3541 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc
     3601 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg
     3661 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca
     3721 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat
     3781 tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga
     3841 tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga
     3901 agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga
     3961 tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag
     4021 tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc
     4081 ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca
     4141 ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggca
     4201 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tccgacgcgc
     4261 tggacgattt cgatctcgac atgctgggtt ctgatgccct cgatgacttt gacctggata
     4321 tgttgggaag cgacgcattg gatgactttg atctggacat gctcggctcc gatgctctgg
     4381 acgatttcga tctcgatatg ttataagaat tcatcgatac cgtcgagatc taacttgttt
     4441 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4501 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4561 tggaggtacc tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     4621 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     4681 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     4741 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     4801 tgtggaaagg acgaaacacc ggagaccacg gcaggtctca gttttagtac tctggaaaca
     4861 gaatctacta aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt
     4921 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4981 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     5041 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     5101 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     5161 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5221 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5281 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5341 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5401 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5461 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5521 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5581 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5641 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5701 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5761 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5821 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5881 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5941 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6001 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6061 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6121 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6181 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6241 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6301 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6361 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6421 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6481 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6541 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6601 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6661 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     6721 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6781 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6841 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6901 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6961 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7021 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7081 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7141 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     7201 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7261 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7321 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7381 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7441 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7501 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7561 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7621 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
