LOCUS       62056_22725        9427 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 9427)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..310
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        337..412
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1380..1665
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1667..1944
                     /label=chicken beta-actin promoter
     CDS             2310..6410
                     /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"
     polyA_signal    6492..6699
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6708..6848
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6923..7378
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7660..7764
                     /label=AmpR promoter
     CDS             7765..8622
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8796..9384
                     /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 gaaacaccgg gtcttcgaga agacctgttt tagagctaga aatagcaagt
      361 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      481 tctgcagaca aatggctcta gagagggcct atttcccatg attccttcat atttgcatat
      541 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      601 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      661 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      721 tttatatatc ttgtggaaag gacgaaacac cggagaccta ttcgccttaa ggtctcggtt
      781 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      841 accgagtcgg tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc
      901 gtttttagcg cgtgcgccaa ttctgcagac aaatggggta ccctccatca ctaggggttc
      961 ctgcggccgc acgcgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     1021 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     1081 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     1141 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     1201 tatcttgtgg aaaggacgaa acaccggaag agcgagctct tctgttttag agctagaaat
     1261 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     1321 ttttttctag aggtaccagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtc
     1381 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1441 acgtcaatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1501 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1561 gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt acatgacctt atgggacttt
     1621 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc
     1681 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     1741 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc
     1801 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
     1861 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
     1921 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     1981 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     2041 agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt
     2101 aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc
     2161 actttttttc aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca
     2221 aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga
     2281 aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg
     2341 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     2401 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     2461 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     2521 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     2581 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     2641 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     2701 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     2761 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     2821 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     2881 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     2941 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     3001 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     3061 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     3121 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3181 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3241 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3301 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     3361 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     3421 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     3481 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     3541 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     3601 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     3661 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     3721 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     3781 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     3841 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     3901 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     3961 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     4021 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     4081 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     4141 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     4201 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     4261 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     4321 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     4381 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     4441 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     4501 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     4561 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     4621 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     4681 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     4741 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     4801 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     4861 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     4921 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     4981 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     5041 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     5101 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     5161 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     5221 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     5281 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     5341 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     5401 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     5461 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     5521 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     5581 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     5641 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     5701 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     5761 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     5821 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     5881 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     5941 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     6001 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     6061 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     6121 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     6181 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     6241 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     6301 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     6361 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg
     6421 cggccacgaa aaaggccggc caggcaaaaa agaaaaagta agaattccta gagctcgctg
     6481 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     6541 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     6601 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     6661 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     6721 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     6781 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     6841 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6901 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6961 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     7021 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     7081 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     7141 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     7201 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     7261 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     7321 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     7381 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     7441 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     7501 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     7561 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     7621 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     7681 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     7741 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     7801 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     7861 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     7921 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     7981 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     8041 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     8101 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     8161 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     8221 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     8281 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     8341 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     8401 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     8461 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     8521 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     8581 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     8641 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     8701 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     8761 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     8821 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     8881 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     8941 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     9001 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     9061 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     9121 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     9181 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     9241 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     9301 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     9361 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     9421 ttttgct
//
