LOCUS       62056_22765        9751 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 9751)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        730..805
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        902..1187
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1189..1466
                     /label=chicken beta-actin promoter
     CDS             1833..5933
                     /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    6798..7005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7014..7154
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      7229..7684
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7966..8070
                     /label=AmpR promoter
     CDS             8071..8928
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      9102..9690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggttcgctc tcacggtccc tgaggtgggc gggcgggccc tggatgacag cgatagaacc
       61 ccggcccgac tcgccctcgc ccccgctctg ggtctgggct tccccagcct agttcacgcc
      121 taggagccgc ctgagcagcc gcgcgcccag cgccacacgc cacgagccct ccccgcctgg
      181 gcgtccccgg atcccgcgag cgctcgggct cccggcttgg aaccagggag gagggaggga
      241 gcgagggagc aaccagctgc ggacccggaa atgccatata agaagcagga aggatccccc
      301 gccggaacaa cccttatttg ggcagcacct tatttggagt ggcccgatat ggcccggccg
      361 cttccggctc tgggaggagg gaagaaggcg gagggagggg caacgcggga actccggagc
      421 tgcgcgggtc ccggaggccc cggcggcggc tagagctcta ggcttccccg aagcctgggc
      481 gcctgggatg cgggcgcggg cgcgggccct agggtgcagg atggaggtgc cgggcgctgt
      541 cggatggggg gcttcacgtc actccgggtc ctcccggccg gtcctgccat attagggctt
      601 cctgcttccc atatatggcc atgtacgtca cgacggaggc ggacccgtgc cgttccagac
      661 ccttcaaata gaggcggatc cggggagtcg cgagagatcc agcgaaacac cgggtcttcg
      721 agaagacctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      781 tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa
      841 taaggctagt ccgtttttag cgcgtgcgcc aattctgcag acaaatggct ctagaggtac
      901 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      961 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1021 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1081 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
     1141 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     1201 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     1261 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
     1321 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
     1381 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     1441 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc
     1501 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
     1561 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
     1621 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
     1681 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
     1741 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
     1801 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
     1861 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1921 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1981 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     2041 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     2101 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     2161 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     2221 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     2281 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     2341 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     2401 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     2461 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     2521 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     2581 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     2641 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     2701 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     2761 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     2821 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     2881 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2941 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     3001 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     3061 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     3121 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     3181 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     3241 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     3301 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     3361 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     3421 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     3481 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     3541 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     3601 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     3661 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     3721 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     3781 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     3841 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3901 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3961 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     4021 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     4081 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     4141 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     4201 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     4261 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     4321 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     4381 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     4441 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     4501 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     4561 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     4621 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     4681 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     4741 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     4801 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     4861 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     4921 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4981 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     5041 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     5101 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     5161 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     5221 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     5281 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     5341 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     5401 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     5461 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     5521 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     5581 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     5641 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     5701 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     5761 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     5821 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     5881 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc
     5941 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg
     6001 gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca gtgagcaagg
     6061 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     6121 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     6181 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     6241 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     6301 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     6361 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     6421 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     6481 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     6541 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     6601 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     6661 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     6721 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggaattc taactagagc
     6781 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     6841 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     6901 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     6961 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     7021 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     7081 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     7141 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     7201 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     7261 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     7321 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     7381 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     7441 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     7501 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     7561 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     7621 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     7681 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     7741 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     7801 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     7861 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     7921 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     7981 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     8041 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     8101 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     8161 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     8221 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     8281 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     8341 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     8401 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     8461 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     8521 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     8581 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     8641 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     8701 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     8761 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     8821 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     8881 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     8941 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     9001 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     9061 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     9121 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     9181 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     9241 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     9301 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     9361 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     9421 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     9481 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     9541 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     9601 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     9661 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     9721 ctggcctttt gctggccttt tgctcacatg t
//
