LOCUS       62056_2000        8976 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 8976)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        179..254
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        351..636
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        638..915
                     /label=chicken beta-actin promoter
     CDS             1282..5382
                     /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             5446..5499
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5500..6096
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    6133..6340
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6349..6489
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6564..7019
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7301..7405
                     /label=AmpR promoter
     CDS             7406..8263
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8437..8976
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
       61 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt ttgaggaatt
      121 tccattgagg aatttccatt gaggaatttc cagaaacacc gggtcttcga gaagacctgt
      181 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      241 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      301 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc cgttacataa
      361 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      421 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      481 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      541 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
      601 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      661 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      721 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg
      781 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
      841 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
      901 agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc
      961 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1021 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg
     1081 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt
     1141 tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat
     1201 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt
     1261 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac
     1321 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     1381 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     1441 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     1501 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     1561 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     1621 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     1681 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     1741 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     1801 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     1861 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     1921 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     1981 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     2041 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     2101 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     2161 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     2221 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     2281 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     2341 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     2401 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     2461 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     2521 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     2581 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     2641 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     2701 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     2761 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     2821 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     2881 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     2941 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     3001 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     3061 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     3121 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     3181 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     3241 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     3301 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     3361 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     3421 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     3481 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     3541 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     3601 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     3661 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     3721 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     3781 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac
     3841 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     3901 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     3961 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     4021 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     4081 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     4141 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     4201 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     4261 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     4321 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     4381 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     4441 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     4501 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     4561 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     4621 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     4681 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     4741 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     4801 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     4861 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     4921 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     4981 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     5041 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     5101 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     5161 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     5221 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     5281 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     5341 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg
     5401 aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca gtggagaggg cagaggaagt
     5461 ctgctaacat gcggtgacgt cgaggagaat cctggcccaa tgaccgagta caagcccacg
     5521 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     5581 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     5641 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     5701 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     5761 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     5821 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     5881 gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     5941 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     6001 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     6061 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag aattctaact agagctcgct
     6121 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     6181 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     6241 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     6301 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag
     6361 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     6421 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     6481 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     6541 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     6601 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     6661 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     6721 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     6781 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     6841 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     6901 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     6961 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     7021 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     7081 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     7141 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     7201 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     7261 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     7321 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     7381 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     7441 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     7501 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     7561 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     7621 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     7681 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     7741 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     7801 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     7861 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     7921 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     7981 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     8041 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     8101 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     8161 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     8221 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     8281 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     8341 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     8401 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     8461 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     8521 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     8581 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     8641 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     8701 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     8761 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     8821 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     8881 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     8941 tctttatagt cctgtcgggt ttcgccacct ctgact
//
