LOCUS       62056_22760        9387 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 9387)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        366..441
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        538..823
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        825..1102
                     /label=chicken beta-actin promoter
     CDS             1469..5569
                     /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    6434..6641
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6650..6790
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6865..7320
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7602..7706
                     /label=AmpR promoter
     CDS             7707..8564
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8738..9326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cactctggcc tctgattggt ccaaggaagg ctggggggca ggacgggagg cgaaaaccct
       61 ggaatattcc cgacctggca gcctcatcga gctcggtgat tggctcagaa gggaaaaggc
      121 gggtctccgt gacgacttat aaaagcccag gggcaagcgg tccggataac ggctagcctg
      181 aggagctgct gcgacagtcc actacctttt tcgagagtga ctcccgttgt cccaaggctt
      241 cccagagcga acctgtgcgg ctgcaggcac cggcgcgtcg agtttccggc gtccggaagg
      301 accgagctct tctcgcggat ccagtgttcc gtttccagag aaacaccggg tcttcgagaa
      361 gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      421 aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag
      481 gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag aggtacccgt
      541 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      601 gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      661 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      721 caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc
      781 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      841 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      901 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg
      961 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
     1021 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     1081 aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct
     1141 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1201 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     1261 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     1321 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     1381 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1441 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1501 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1561 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1621 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1681 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1741 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1801 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1861 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1921 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1981 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     2041 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     2101 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     2161 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     2221 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     2281 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     2341 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     2401 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     2461 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     2521 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     2581 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2641 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2701 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2761 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2821 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2881 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2941 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     3001 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     3061 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     3121 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     3181 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     3241 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     3301 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     3361 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     3421 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     3481 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     3541 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     3601 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     3661 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     3721 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     3781 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     3841 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     3901 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3961 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     4021 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     4081 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     4141 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     4201 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     4261 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     4321 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     4381 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     4441 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     4501 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     4561 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     4621 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     4681 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     4741 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     4801 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     4861 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     4921 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     4981 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     5041 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     5101 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     5161 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     5221 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     5281 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     5341 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     5401 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     5461 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     5521 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc
     5581 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag
     5641 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga gcaagggcga
     5701 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     5761 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     5821 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     5881 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     5941 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     6001 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     6061 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     6121 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     6181 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     6241 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     6301 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     6361 cgccgccggg atcactctcg gcatggacga gctgtacaag gaattctaac tagagctcgc
     6421 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     6481 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     6541 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     6601 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
     6661 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     6721 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     6781 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     6841 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     6901 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     6961 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     7021 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     7081 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     7141 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     7201 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     7261 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     7321 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     7381 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     7441 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     7501 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     7561 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     7621 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     7681 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     7741 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     7801 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     7861 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     7921 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     7981 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     8041 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     8101 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     8161 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     8221 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     8281 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     8341 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     8401 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     8461 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     8521 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     8581 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     8641 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     8701 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     8761 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     8821 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     8881 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     8941 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     9001 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     9061 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     9121 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     9181 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     9241 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     9301 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     9361 ccttttgctg gccttttgct cacatgt
//
