LOCUS Exported 8697 bp ds-DNA circular SYN 04-DEC-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..8697
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 8..293
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 295..572
/label=chicken beta-actin promoter
intron 573..800
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 812..821
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 821..886
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 893..913
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 938..5038
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/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 5039..5086
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5120..5327
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 5336..5476
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5551..6006
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS 6627..7418
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=Spe
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
rep_origin 7601..8189
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 8267..8507
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 8534..8609
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
ORIGIN
1 tgagacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
421 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
601 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
661 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
721 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
781 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg
841 agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa
901 gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct
961 ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
1021 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
1081 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
1141 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
1201 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
1261 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
1321 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
1381 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
1441 gatcgagggc gacctgaacc ccgacaacag cgacgttgac aagctgttca tccagctggt
1501 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
1561 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
1621 gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac
1681 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
1741 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
1801 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
1861 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
1921 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
1981 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
2041 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
2101 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
2161 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
2221 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
2281 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
2341 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
2401 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
2461 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
2521 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
2581 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
2641 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
2701 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
2761 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
2821 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
2881 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
2941 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
3001 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
3061 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
3121 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
3181 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
3241 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
3301 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
3361 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
3421 catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga
3481 cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa
3541 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
3601 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
3661 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
3721 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
3781 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
3841 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
3901 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
3961 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
4021 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
4081 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
4141 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
4201 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
4261 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
4321 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
4381 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
4441 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
4501 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
4561 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
4621 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
4681 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
4741 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
4801 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
4861 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
4921 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
4981 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa
5041 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagtaag aattcctaga
5101 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
5161 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
5221 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
5281 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa
5341 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
5401 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
5461 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
5521 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
5581 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
5641 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
5701 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
5761 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
5821 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
5881 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
5941 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
6001 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
6061 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
6121 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
6181 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
6241 taataatggt ttcttagacg tcaggtggca cttccagcca ggacagaaat gcctcgactt
6301 cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc
6361 agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
6421 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
6481 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
6541 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
6601 gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc
6661 aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
6721 atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg
6781 ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg
6841 aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg
6901 tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat
6961 ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
7021 ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg
7081 aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc
7141 tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
7201 gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg
7261 caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc
7321 ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact
7381 acgtgaaagg cgagatcacc aaggtagtcg gcaaataaaa gcattggtaa ctgtcagacc
7441 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
7501 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
7561 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
7621 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
7681 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
7741 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
7801 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
7861 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
7921 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
7981 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
8041 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
8101 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
8161 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
8221 tggccttttg ctggcctttt gctcaggtct cactctagag gcatgtgagg gcctatttcc
8281 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
8341 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
8401 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
8461 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc
8521 ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
8581 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
8641 aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctcta
//