LOCUS       62056_22710        9279 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9279)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          208..435
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             573..4673
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /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             4785..5501
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    5529..5736
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5745..5885
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5960..6415
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6697..6801
                     /label=AmpR promoter
     CDS             6802..7659
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7833..8421
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        8483..8723
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        8750..8825
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8922..9207
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        9209..9279
                     /label=chicken beta-actin promoter
ORIGIN
        1 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca
       61 ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc
      121 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc
      181 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc
      241 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      301 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
      361 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
      421 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
      481 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
      541 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
      601 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
      661 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
      721 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
      781 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
      841 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
      901 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
      961 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1021 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1081 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1141 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1201 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     1261 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     1321 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     1381 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     1441 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     1501 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     1561 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     1621 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     1681 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     1741 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     1801 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     1861 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     1921 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     1981 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2041 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2101 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     2161 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     2221 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     2281 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     2341 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     2401 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     2461 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     2521 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     2581 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     2641 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     2701 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     2761 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     2821 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     2881 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     2941 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3001 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3061 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3121 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3181 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     3241 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     3301 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     3361 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     3421 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     3481 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     3541 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     3601 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     3661 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     3721 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     3781 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     3841 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     3901 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     3961 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     4021 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     4081 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     4141 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     4201 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     4261 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     4321 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     4381 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     4441 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     4501 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     4561 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     4621 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc
     4681 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcgca acaaacttct
     4741 ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgatggtg tctaagggcg
     4801 aagagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     4861 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     4921 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     4981 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     5041 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     5101 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     5161 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     5221 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     5281 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     5341 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     5401 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     5461 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaactagag ctcgctgatc
     5521 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     5581 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     5641 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     5701 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat
     5761 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     5821 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     5881 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     5941 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     6001 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     6061 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     6121 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     6181 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     6241 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     6301 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     6361 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     6421 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     6481 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     6541 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     6601 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     6661 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6721 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6781 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6841 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6901 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6961 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     7021 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7081 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7141 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     7201 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     7261 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     7321 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     7381 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     7441 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7501 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     7561 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7621 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7681 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7741 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7801 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7861 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7921 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7981 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     8041 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     8101 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     8161 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     8221 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     8281 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     8341 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8401 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8461 tgctggcctt ttgctcacat gtgagggcct atttcccatg attccttcat atttgcatat
     8521 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     8581 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     8641 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     8701 tttatatatc ttgtggaaag gacgaaacac cgggtcttcg agaagacctg ttttagagct
     8761 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     8821 ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt ccgtttttag
     8881 cgcgtgcgcc aattctgcag acaaatggct ctagaggtac ccgttacata acttacggta
     8941 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca
     9001 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     9061 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     9121 cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     9181 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     9241 cccatctccc ccccctcccc acccccaatt ttgtattta
//
