LOCUS       Exported                9243 bp ds-DNA     circular SYN 23-JUL-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 9243)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jul 31, 2019 from SnapGene Viewer 4.3.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9243
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        62..302
                     /label=U6 promoter
     primer_bind     107..131
                     /label=pORANGE sequencing primer FW
     misc_feature    311..331
                     /label=Target sequence
     misc_RNA        332..407
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    408..418
                     /label=Termination sequence
                     /note="RNA polymerase will stop transcription at this 
                     position and fall off"
     misc_feature    503..505
                     /label=PAM
     misc_feature    506..525
                     /label=Target sequence
     CDS             526..1239
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    1241..1243
                     /label=PAM
     misc_feature    1244..1263
                     /label=Target sequence
     enhancer        1308..1593
                     /label=CMV enhancer
     primer_bind     complement(1316..1335)
                     /label=pORANGE sequencing primer RV
     promoter        1595..1872
                     /label=chicken beta-actin promoter
     intron          1873..2100
                     /label=hybrid intron
     CDS             2118..2120
                     /codon_start=1
                     /label=START
                     /translation="M"
     CDS             2121..2147
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2151..2171
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2181..6281
                     /codon_start=1
                     /product="Cas9 is from HITI construct. Suzuki et al., 2016"
                     /label=Cas9
                     /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             6285..6305
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6351..6558
                     /label=bGH poly(A) signal
     repeat_region   6567..6707
                     /label=AAV2 ITR
     rep_origin      6782..7237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 stays for filamentous phages. It initiates a 
                     ""rolling circle"" replication in phage-infected bacteria 
                     and is useful if there is a need to produce vector as a 
                     single-stranded DNA"
     promoter        7519..7623
                     /label=AmpR promoter
     CDS             7624..8484
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8655..9243
                     /direction=RIGHT
                     /label=ori
                     /note="Ori for replication in E.coli"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      121 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      181 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
      241 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
      301 acgaaacacc gcttcaacac cctgccgtcc agttttagag ctagaaatag caagttaaaa
      361 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc
      481 agacaaatgg ctctagaagc ttccctggac ggcagggtgt tgaaggtgag caagggcgag
      541 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      601 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      661 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
      721 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
      781 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      841 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      901 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
      961 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1021 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1081 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1141 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1201 gccgccggga tcactctcgg catggacgag ctgtacaaga ccctggacgg cagggtgttg
     1261 aagctcgagg atcctaggac gcgtcctagg acgcgtccag aggtacccgt tacataactt
     1321 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaatagta
     1381 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     1441 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     1501 aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc tacttggcag
     1561 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
     1621 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
     1681 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
     1741 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
     1801 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
     1861 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
     1921 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
     1981 ggcccttctc ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg
     2041 gttggtgggg tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag
     2101 gttggaccgg tgccaccatg tacccatacg atgttccaga ttacgcttcg ccgaagaaaa
     2161 agcgcaaggt cgaagcgtcc gacaagaagt acagcatcgg cctggacatc ggcaccaact
     2221 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
     2281 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca
     2341 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
     2401 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
     2461 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
     2521 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
     2581 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
     2641 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
     2701 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     2761 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     2821 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     2881 atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     2941 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     3001 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     3061 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     3121 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     3181 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     3241 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt
     3301 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca
     3361 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     3421 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     3481 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     3541 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     3601 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
     3661 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     3721 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     3781 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga
     3841 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     3901 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     3961 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     4021 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     4081 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     4141 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     4201 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     4261 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     4321 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     4381 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     4441 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     4501 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     4561 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     4621 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     4681 actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca
     4741 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     4801 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga
     4861 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     4921 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     4981 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag
     5041 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     5101 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
     5161 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg
     5221 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     5281 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     5341 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg
     5401 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg
     5461 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     5521 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     5581 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     5641 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     5701 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     5761 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     5821 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     5881 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
     5941 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg
     6001 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca
     6061 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc
     6121 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
     6181 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
     6241 tgtacgagac acggatcgac ctgtctcagc tgggaggcga cagccccaag aagaagagaa
     6301 aggtggaggc cagctaagaa ttctaactag agctcgctga tcagcctcga ctgtgccttc
     6361 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     6421 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     6481 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa
     6541 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc
     6601 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     6661 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     6721 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     6781 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     6841 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     6901 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     6961 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     7021 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     7081 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     7141 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     7201 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     7261 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     7321 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     7381 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     7441 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     7501 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     7561 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     7621 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     7681 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     7741 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     7801 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     7861 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     7921 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     7981 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     8041 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     8101 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     8161 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     8221 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     8281 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     8341 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     8401 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     8461 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     8521 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     8581 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     8641 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     8701 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     8761 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     8821 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     8881 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     8941 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     9001 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     9061 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     9121 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     9181 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     9241 aaa
//
