LOCUS       62056_22770        9325 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 9325)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        476..761
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        763..1040
                     /label=chicken beta-actin promoter
     CDS             1407..5507
                     /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    6372..6579
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6588..6728
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6803..7258
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7540..7644
                     /label=AmpR promoter
     CDS             7645..8502
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8676..9264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agagtcgaca ctagtctcga gatcgataag cttcctagag gtacccgtta
      481 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      541 caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      601 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      661 atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg gactttccta
      721 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
      781 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
      841 tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg
      901 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga
      961 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
     1021 agcgaagcgc gcggcgggcg ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc
     1081 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     1141 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
     1201 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
     1261 ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga gactacaagg
     1321 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
     1381 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gacatcggca
     1441 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     1501 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     1561 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     1621 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     1681 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     1741 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     1801 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     1861 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     1921 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     1981 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     2041 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     2101 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
     2161 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     2221 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     2281 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     2341 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     2401 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     2461 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     2521 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     2581 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     2641 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     2701 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     2761 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     2821 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     2881 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     2941 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     3001 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     3061 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     3121 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     3181 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     3241 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     3301 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     3361 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     3421 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     3481 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     3541 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     3601 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     3661 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     3721 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     3781 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     3841 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     3901 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
     3961 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     4021 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     4081 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     4141 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     4201 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     4261 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     4321 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
     4381 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     4441 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     4501 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     4561 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     4621 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     4681 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
     4741 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     4801 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     4861 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     4921 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     4981 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     5041 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     5101 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     5161 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     5221 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     5281 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     5341 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
     5401 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     5461 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg
     5521 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag
     5581 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc aagggcgagg
     5641 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     5701 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     5761 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     5821 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     5881 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     5941 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     6001 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     6061 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     6121 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     6181 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     6241 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     6301 ccgccgggat cactctcggc atggacgagc tgtacaagga attctaacta gagctcgctg
     6361 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     6421 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     6481 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     6541 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     6601 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     6661 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     6721 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6781 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6841 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     6901 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6961 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     7021 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     7081 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     7141 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     7201 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     7261 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     7321 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     7381 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     7441 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     7501 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     7561 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     7621 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     7681 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     7741 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     7801 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     7861 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     7921 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     7981 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     8041 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     8101 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     8161 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     8221 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     8281 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     8341 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     8401 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     8461 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     8521 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     8581 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     8641 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     8701 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     8761 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     8821 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     8881 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     8941 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     9001 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     9061 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     9121 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     9181 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     9241 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     9301 ttttgctggc cttttgctca catgt
//
