LOCUS       62056_1036        9227 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 9227)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             827..4927
                     /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             5036..5701
                     /codon_start=1
                     /label=OFP
                     /note="orange fluorescent protein from Cerianthus (Ip et
                     al., 2007)"
                     /translation="MNLSKNVSVSVYMKGNVNNHEFEYDGEGGGDPYTGKYSMKMTLRG
                     QNSLPFSYDIITTAFQYGFRVFTKYPEGIVDYFKDSLPDAFQWNRRIVFEDGGVLNMSS
                     DITYKDNVLHGDVKAEGVNFPPNGPVMKNEIVMEEPTEETFTPKNGVLVGFCPKAYLLK
                     DGSYYYGNMTTFYRSKKSGQAPPGYHFVKHRLVKTNVGHGFKTVEQTEYATAHVSDLPK
                     "
     polyA_signal    5785..5833
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6035..6463
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6483..6723
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        6760..6835
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(6885..6901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6909..6925)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6933..6963)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6978..6999)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(7287..7875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8049..8906)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8907..9011)
                     /label=AmpR promoter
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 agcggccgcc accatgggca agcccatccc taaccccctg ttggggctgg acagcaccgc
      781 tcccaaaaag aaaaggaagg tgggcattca cggcgtgcct gcggccgaca aaaagtacag
      841 catcggcctt gatatcggca ccaatagcgt gggctgggcc gttatcacag acgaatacaa
      901 ggtacccagc aagaagttca aggtgctggg gaatacagac aggcactcta tcaagaaaaa
      961 ccttatcggg gctctgctgt ttgactcagg cgagaccgcc gaggccacca ggttgaagag
     1021 gaccgcaagg cgaaggtaca cccggaggaa gaacaggatc tgctatctgc aggagatctt
     1081 cagcaacgag atggccaagg tggacgacag cttcttccac aggctggagg agagcttcct
     1141 tgtcgaggag gataagaagc acgaacgaca ccccatcttc ggcaacatag tcgacgaggt
     1201 cgcttatcac gagaagtacc ccaccatcta ccacctgcga aagaaattgg tggatagcac
     1261 cgataaagcc gacttgcgac ttatctactt ggctctggcg cacatgatta agttcagggg
     1321 ccacttcctg atcgagggcg accttaaccc cgacaacagt gacgtagaca aattgttcat
     1381 ccagcttgta cagacctata accagctgtt cgaggaaaac cctattaacg ccagcggggt
     1441 ggatgcgaag gccatactta gcgccaggct gagcaaaagc aggcgcttgg agaacctgat
     1501 agcccagctg cccggtgaaa agaagaacgg cctcttcggt aatctgattg ccctgagcct
     1561 gggcctgacc cccaacttca agagcaactt cgacctggca gaagatgcca agctgcagtt
     1621 gagtaaggac acctatgacg acgacttgga caatctgctc gcccaaatcg gcgaccagta
     1681 cgctgacctg ttcctcgccg ccaagaacct ttctgacgca atcctgctta gcgatatcct
     1741 tagggtgaac acagagatca ccaaggcccc cctgagcgcc agcatgatca agaggtacga
     1801 cgagcaccat caggacctga cccttctgaa ggccctggtg aggcagcaac tgcccgagaa
     1861 gtacaaggag atctttttcg accagagcaa gaacggctac gccggctaca tcgacggcgg
     1921 agccagccaa gaggagttct acaagttcat caagcccatc ctggagaaga tggatggcac
     1981 cgaggagctg ctggtgaagc tgaacaggga agatttgctc cggaagcaga ggacctttga
     2041 caacggtagc atcccccacc agatccacct gggcgagctg cacgcaatac tgaggcgaca
     2101 ggaggatttc taccccttcc tcaaggacaa tagggagaaa atcgaaaaga ttctgacctt
     2161 caggatcccc tactacgtgg gccctcttgc caggggcaac agccgattcg cttggatgac
     2221 aagaaagagc gaggagacca tcaccccctg gaacttcgag gaagtggtgg acaaaggagc
     2281 aagcgcgcag tctttcatcg aacggatgac caatttcgac aaaaacctgc ctaacgagaa
     2341 ggtgctgccc aagcacagcc tgctttacga gtacttcacc gtgtacaacg agctcaccaa
     2401 ggtgaaatat gtgaccgagg gcatgcgaaa acccgctttc ctgagcggcg agcagaagaa
     2461 ggccatcgtg gacctgctgt tcaagaccaa caggaaggtg accgtgaagc agctgaagga
     2521 ggactacttc aagaagatcg agtgctttga tagcgtggaa ataagcggcg tggaggacag
     2581 gttcaacgcc agcctgggca cctaccacga cttgttgaag ataatcaaag acaaggattt
     2641 cctggataat gaggagaacg aggatatact cgaggacatc gtgctgactt tgaccctgtt
     2701 tgaggaccga gagatgattg aagaaaggct caaaacctac gcccacctgt tcgacgacaa
     2761 agtgatgaaa caactgaaga gacgaagata caccggctgg ggcagactgt ccaggaagct
     2821 catcaacggc attagggaca agcagagcgg caagaccatc ctggatttcc tgaagtccga
     2881 cggcttcgcc aaccgaaact tcatgcagct gattcacgat gacagcttga ccttcaagga
     2941 ggacatccag aaggcccagg ttagcggcca gggcgactcc ctgcacgaac atattgcaaa
     3001 cctggcaggc tcccctgcga tcaagaaggg catactgcag accgttaagg ttgtggacga
     3061 attggtcaag gtcatgggca ggcacaagcc cgaaaacata gttatagaga tggccagaga
     3121 gaaccagacc acccaaaagg gccagaagaa cagccgggag cgcatgaaaa ggatcgagga
     3181 gggtatcaag gaactcggaa gccagatcct caaagagcac cccgtggaga atacccagct
     3241 ccagaacgag aagctgtacc tgtactacct gcagaacggc agggacatgt acgttgacca
     3301 ggagttggac atcaacaggc tttcagacta tgacgtggat cacatagtgc cccagagctt
     3361 tcttaaagac gatagcatcg acaacaaggt cctgacccgc tccgacaaaa acaggggcaa
     3421 aagcgacaac gtgccaagcg aagaggtggt taaaaagatg aagaactact ggaggcaact
     3481 gctcaacgcg aaattgatca cccagagaaa gttcgataac ctgaccaagg ccgagagggg
     3541 cggactctcc gaacttgaca aagcgggctt cataaagagg cagctggtcg agacccgaca
     3601 gatcacgaag cacgtggccc aaatcctcga cagcagaatg aataccaagt acgatgagaa
     3661 tgacaaactc atcagggaag tgaaagtgat taccctgaag agcaagttgg tgtccgactt
     3721 tcgcaaagat ttccagttct acaaggtgag ggagatcaac aactaccacc atgcccacga
     3781 cgcatacctg aacgccgtgg tcggcaccgc cctgattaag aagtatccaa agctggagtc
     3841 cgaatttgtc tacggcgact acaaagttta cgatgtgagg aagatgatcg ctaagagcga
     3901 acaggagatc ggcaaggcca ccgctaagta tttcttctac agcaacatca tgaacttttt
     3961 caagaccgag atcacacttg ccaacggcga aatcaggaag aggccgctta tcgagaccaa
     4021 cggtgagacc ggcgagatcg tgtgggacaa gggcagggac ttcgccaccg tgaggaaagt
     4081 cctgagcatg ccccaggtga atattgtgaa aaaaactgag gtgcagacag gcggctttag
     4141 caaggaatcc atcctgccca agaggaacag cgacaagctg atcgcccgga agaaggactg
     4201 ggaccctaag aagtatggag gcttcgacag ccccaccgta gcctacagcg tgctggtggt
     4261 cgcgaaggta gagaagggga agagcaagaa actgaagagc gtgaaggagc tgctcggcat
     4321 aaccatcatg gagaggtcca gctttgagaa gaaccccatt gactttttgg aagccaaggg
     4381 ctacaaagag gtcaaaaagg acctgatcat caaactcccc aagtactccc tgtttgaatt
     4441 ggagaacggc agaaagagga tgctggcgag cgctggggaa ctgcaaaagg gcaacgaact
     4501 ggcgctgccc agcaagtacg tgaattttct gtacctggcg tcccactacg aaaagctgaa
     4561 aggcagcccc gaggacaacg agcagaagca gctgttcgtg gagcagcaca agcattacct
     4621 ggacgagata atcgagcaaa tcagcgagtt cagcaagagg gtgattctgg ccgacgcgaa
     4681 cctggataag gtcctcagcg cctacaacaa gcaccgagac aaacccatca gggagcaggc
     4741 cgagaatatc atacacctgt tcaccctgac aaatctgggc gcacctgcgg cattcaaata
     4801 cttcgatacc accatcgaca ggaaaaggta cactagcact aaggaggtgc tggatgccac
     4861 cttgatccac cagtccatta ccggcctgta tgagaccagg atcgacctga gccagcttgg
     4921 aggcgactct agggcggacc caaaaaagaa aaggaaggtg gaattctcta gaggcagtgg
     4981 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatgaa
     5041 cctgagcaaa aacgtgagcg tgagcgtgta tatgaagggg aacgtcaaca atcatgagtt
     5101 tgagtacgac ggggaaggtg gtggtgatcc ttatacaggt aaatattcca tgaagatgac
     5161 gctacgtggt caaaattccc tacccttttc ctatgatatc attaccacgg catttcagta
     5221 tggtttccgc gtatttacaa aataccctga gggaattgtt gactatttta aggactcgct
     5281 tcccgacgca ttccagtgga acagacgaat tgtgtttgaa gatggtggag tactaaacat
     5341 gagcagtgat atcacatata aagataatgt tctgcatggt gacgtcaagg ctgagggagt
     5401 gaacttcccg ccgaatgggc cagtgatgaa gaatgaaatt gtgatggagg aaccgactga
     5461 agaaacattt actccaaaaa acggggttct tgttggcttt tgtcccaaag cgtacttact
     5521 taaagacggt tcctattact atggaaatat gacaacattt tacagatcca agaaatctgg
     5581 ccaggcacct cctgggtatc actttgttaa gcatcgtctc gtcaagacca atgtgggaca
     5641 tggatttaag acggttgagc agactgaata tgccactgct catgtcagtg atcttcccaa
     5701 gttcgaagct tgataatgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa
     5761 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt
     5821 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     5881 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     5941 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagca
     6001 gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     6061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6241 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6301 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6361 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6421 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattaag gtcgggcagg
     6481 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     6541 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     6601 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     6661 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     6721 gacgaaacac cgtcaagagg aacaagcttt actcaagagg ttttagagct agaaatagca
     6781 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     6841 tctagtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     6901 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     6961 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     7021 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     7081 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     7141 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     7201 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     7261 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     7321 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     7381 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     7441 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     7501 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     7561 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     7621 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     7681 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     7741 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     7801 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     7861 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     7921 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     7981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     8041 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     8101 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     8161 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     8221 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     8281 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     8341 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     8401 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     8461 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     8521 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     8581 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     8641 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     8701 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     8761 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     8821 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     8881 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     8941 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     9001 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     9061 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     9121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     9181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagg
//
