LOCUS       62056_2275        9158 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 9158)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             882..4982
                     /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             5100..5816
                     /codon_start=1
                     /label=mCerulean
                     /note="enhanced monomeric variant of CFP (Rizzo et al.,
                     2004)"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    5848..6055
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6063..6303
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        6332..6407
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   6421..6561
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6636..7091
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7373..7477
                     /label=AmpR promoter
     CDS             7478..8335
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8509..9097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggactat aaggaccacg
      781 acggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa
      841 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
      901 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
      961 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     1021 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     1081 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     1141 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     1201 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     1261 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     1321 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1381 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1441 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     1501 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     1561 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     1621 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     1681 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     1741 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     1801 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     1861 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     1921 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     1981 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     2041 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     2101 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     2161 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     2221 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     2281 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     2341 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2401 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     2461 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     2521 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     2581 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     2641 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     2701 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     2761 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     2821 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     2881 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     2941 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     3001 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     3061 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     3121 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3181 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     3241 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     3301 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3361 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     3421 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     3481 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     3541 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     3601 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     3661 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     3721 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     3781 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     3841 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     3901 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     3961 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4021 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     4081 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     4141 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     4201 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     4261 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     4321 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     4381 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     4441 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     4501 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     4561 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     4621 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     4681 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     4741 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     4801 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     4861 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     4921 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     4981 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gagttcggca
     5041 gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccaa
     5101 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     5161 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     5221 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     5281 tcgtgaccac cctgacctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc
     5341 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     5401 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     5461 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     5521 agctggagta caacgccatc agcgacaacg tctatatcac cgccgacaag cagaagaacg
     5581 gcatcaaggc caacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     5641 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     5701 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     5761 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtgaa
     5821 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     5881 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5941 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     6001 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac
     6061 ctgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     6121 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     6181 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     6241 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     6301 gacgaaacac cggagaccac ggcaggtctc agttttagta ctctggaaac agaatctact
     6361 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc
     6421 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     6481 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     6541 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     6601 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     6661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     6721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     6781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     6841 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     6901 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6961 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     7021 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     7081 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7141 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     7201 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     7261 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     7321 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     7381 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     7441 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     7501 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     7561 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     7621 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     7681 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     7741 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     7801 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     7861 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     7921 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     7981 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     8041 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     8101 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     8161 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     8221 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     8281 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     8341 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     8401 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     8461 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     8521 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     8581 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     8641 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     8701 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     8761 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     8821 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     8881 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     8941 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     9001 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     9061 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     9121 acggttcctg gccttttgct ggccttttgc tcacatgt
//
