LOCUS Exported 10239 bp ds-DNA circular SYN 07-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..10239
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 78..666
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_RNA 804..879
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 1120..5226
/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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 6793..7392
/codon_start=1
/label=repC
/translation="MSENVIKETENKKNSRGRNWTFVLYPESAKAEWLEYLKELHIQFV
VSPLHDRDTDTEDRMKKEHYHILVMYEGNKSYEQIKIITEELNATIPQIAGSVKGLVRY
MLHMDDPNKFKYQKEDMIVYGGVDVDELLKKTTTDRYKLIKEMIEFIDEQGIVEFKSLM
DYAMKFKFDDWFPLLCDNSAYVIQEYIKSNRYKSDR"
CDS 8288..8938
/codon_start=1
/label=Chl
/translation="MKFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQELSDRPNDWLL"
CDS 9311..10105
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Kan
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 10136..10230
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
ORIGIN
1 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
61 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
121 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
181 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
241 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
301 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
361 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
421 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
481 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
541 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
601 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
661 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
721 cacatgttct ttcctgcgtt gtcgacagag tttgcaaaat atacagggga ttatatataa
781 tggaaacgag accattggtc tcagttttag agctagaaat agcaagttaa aataaggcta
841 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaga tctgtccata
901 cccatggtct agaatgctcg agtcagaaaa atatacctgt atcttttttc aaaagcaaac
961 atgctttggg taaacatgta ggtattaacg tcaatgcgac aatagtagca ttgattaaat
1021 gagaattagt aagtgtttta cttactaaat tttatttaac ctaaaaatga accacctgga
1081 tgtgtgggat taaaaagtga agagaggagg acatatcaca tggataagaa atactcaata
1141 ggcttagata tcggcacaaa tagcgtcgga tgggcggtga tcactgatga atataaggtt
1201 ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc acagtatcaa aaaaaatctt
1261 ataggggctc ttttatttga cagtggagag acagcggaag cgactcgtct caaacggaca
1321 gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt atctacagga gattttttca
1381 aatgagatgg cgaaagtaga tgatagtttc tttcatcgac ttgaagagtc ttttttggtg
1441 gaagaagaca agaagcatga acgtcatcct atttttggaa atatagtaga tgaagttgct
1501 tatcatgaga aatatccaac tatctatcat ctgcgaaaaa aattggtaga ttctactgat
1561 aaagcggatt tgcgcttaat ctatttggcc ttagcgcata tgattaagtt tcgtggtcat
1621 tttttgattg agggagattt aaatcctgat aatagtgatg tggacaaact atttatccag
1681 ttggtacaaa cctacaatca attatttgaa gaaaacccta ttaacgcaag tggagtagat
1741 gctaaagcga ttctttctgc acgattgagt aaatcaagac gattagaaaa tctcattgct
1801 cagctccccg gtgagaagaa aaatggctta tttgggaatc tcattgcttt gtcattgggt
1861 ttgaccccta attttaaatc aaattttgat ttggcagaag atgctaaatt acagctttca
1921 aaagatactt acgatgatga tttagataat ttattggcgc aaattggaga tcaatatgct
1981 gatttgtttt tggcagctaa gaatttatca gatgctattt tactttcaga tatcctaaga
2041 gtaaatactg aaataactaa ggctccccta tcagcttcaa tgattaaacg ctacgatgaa
2101 catcatcaag acttgactct tttaaaagct ttagttcgac aacaacttcc agaaaagtat
2161 aaagaaatct tttttgatca atcaaaaaac ggatatgcag gttatattga tgggggagct
2221 agccaagaag aattttataa atttatcaaa ccaattttag aaaaaatgga tggtactgag
2281 gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca agcaacggac ctttgacaac
2341 ggctctattc cccatcaaat tcacttgggt gagctgcatg ctattttgag aagacaagaa
2401 gacttttatc catttttaaa agacaatcgt gagaagattg aaaaaatctt gacttttcga
2461 attccttatt atgttggtcc attggcgcgt ggcaatagtc gttttgcatg gatgactcgg
2521 aagtctgaag aaacaattac cccatggaat tttgaagaag ttgtcgataa aggtgcttca
2581 gctcaatcat ttattgaacg catgacaaac tttgataaaa atcttccaaa tgaaaaagta
2641 ctaccaaaac atagtttgct ttatgagtat tttacggttt ataacgaatt gacaaaggtc
2701 aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc
2761 attgttgatt tactcttcaa aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat
2821 tatttcaaaa aaatagaatg ttttgatagt gttgaaattt caggagttga agatagattt
2881 aatgcttcat taggtaccta ccatgatttg ctaaaaatta ttaaagataa agattttttg
2941 gataatgaag aaaatgaaga tatcttagag gatattgttt taacattgac cttatttgaa
3001 gatagggaga tgattgagga aagacttaaa acatatgctc acctctttga tgataaggtg
3061 atgaaacagc ttaaacgtcg ccgttatact ggttggggac gtttgtctcg aaaattgatt
3121 aatggtatta gggataagca atctggcaaa acaatattag attttttgaa atcagatggt
3181 tttgccaatc gcaattttat gcagctgatc catgatgata gtttgacatt taaagaagac
3241 attcaaaaag cacaagtgtc tggacaaggc gatagtttac atgaacatat tgcaaattta
3301 gctggtagcc ctgctattaa aaaaggtatt ttacagactg taaaagttgt tgatgaattg
3361 gtcaaagtaa tggggcggca taagccagaa aatatcgtta ttgaaatggc acgtgaaaat
3421 cagacaactc aaaagggcca gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt
3481 atcaaagaat taggaagtca gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa
3541 aatgaaaagc tctatctcta ttatctccaa aatggaagag acatgtatgt ggaccaagaa
3601 ttagatatta atcgtttaag tgattatgat gtcgatcaca ttgttccaca aagtttcctt
3661 aaagacgatt caatagacaa taaggtctta acgcgttctg ataaaaatcg tggtaaatcg
3721 gataacgttc caagtgaaga agtagtcaaa aagatgaaaa actattggag acaacttcta
3781 aacgccaagt taatcactca acgtaagttt gataatttaa cgaaagctga acgtggaggt
3841 ttgagtgaac ttgataaagc tggttttatc aaacgccaat tggttgaaac tcgccaaatc
3901 actaagcatg tggcacaaat tttggatagt cgcatgaata ctaaatacga tgaaaatgat
3961 aaacttattc gagaggttaa agtgattacc ttaaaatcta aattagtttc tgacttccga
4021 aaagatttcc aattctataa agtacgtgag attaacaatt accatcatgc ccatgatgcg
4081 tatctaaatg ccgtcgttgg aactgctttg attaagaaat atccaaaact tgaatcggag
4141 tttgtctatg gtgattataa agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa
4201 gaaataggca aagcaaccgc aaaatatttc ttttactcta atatcatgaa cttcttcaaa
4261 acagaaatta cacttgcaaa tggagagatt cgcaaacgcc ctctaatcga aactaatggg
4321 gaaactggag aaattgtctg ggataaaggg cgagattttg ccacagtgcg caaagtattg
4381 tccatgcccc aagtcaatat tgtcaagaaa acagaagtac agacaggcgg attctccaag
4441 gagtcaattt taccaaaaag aaattcggac aagcttattg ctcgtaaaaa agactgggat
4501 ccaaaaaaat atggtggttt tgatagtcca acggtagctt attcagtcct agtggttgct
4561 aaggtggaaa aagggaaatc gaagaagtta aaatccgtta aagagttact agggatcaca
4621 attatggaaa gaagttcctt tgaaaaaaat ccgattgact ttttagaagc taaaggatat
4681 aaggaagtta aaaaagactt aatcattaaa ctacctaaat atagtctttt tgagttagaa
4741 aacggtcgta aacggatgct ggctagtgcc ggagaattac aaaaaggaaa tgagctggct
4801 ctgccaagca aatatgtgaa ttttttatat ttagctagtc attatgaaaa gttgaagggt
4861 agtccagaag ataacgaaca aaaacaattg tttgtggagc agcataagca ttatttagat
4921 gagattattg agcaaatcag tgaattttct aagcgtgtta ttttagcaga tgccaattta
4981 gataaagttc ttagtgcata taacaaacat agagacaaac caatacgtga acaagcagaa
5041 aatattattc atttatttac gttgacgaat cttggagctc ccgctgcttt taaatatttt
5101 gatacaacaa ttgatcgtaa acgatatacg tctacaaaag aagttttaga tgccactctt
5161 atccatcaat ccatcactgg tctttatgaa acacgcattg atttgagtca gctaggaggt
5221 gactgatggc tggttggcgt actgttgtgg taaataccca tgggtatgga cagttttccc
5281 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
5341 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
5401 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
5461 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
5521 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
5581 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
5641 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
5701 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc
5761 ctgcagaacg gattgttgat gattacgaaa atattaagag cacagactat tacacagaaa
5821 atcaagaatt aaaaaaacgt agagagagtt tgaaagaagt agtgaataca tggaaagagg
5881 ggtatcacga aaaaagtaaa gaggttaata aattaaagcg agagaatgat agtttgaatg
5941 agcagttgaa tgtatcagag aaatttcaag atagtacagt gactttatat cgtgctgcga
6001 gggcgaattt ccctgggttt gagaaagggt ttaataggct taaagagaaa ttctttaatg
6061 attccaaatt cgagcgtgtg ggacagttta tggatgttgt acaggataat gtccagaagg
6121 tcgatagaaa gcgtgagaaa cagcgtacag acgatttaga gatgtagagg tacttttatg
6181 ccgagaaaac tttttgcgtg tgacagtcct taaaatatac ttagagcgta agcgaaagta
6241 gtagcgacag ctattaactt tcggttgcaa agctctagga tttttaatgg acgcagcgca
6301 tcacacgcaa aaaggaaatt ggaataaatg cgaaatttga gatgttaatt aaagaccttt
6361 ttgaggtctt tttttcttag atttttgggg ttatttaggg gagaaaacat aggggggtac
6421 tacgacctcc cccctaggtg tccattgtcc attgtccaaa caaataaata aatattgggt
6481 ttttaatgtt aaaaggttgt tttttatgtt aaagtgaaaa aaacagatgt tgggaggtac
6541 agtgatggtt gtagatagaa aagaagagaa aaaagttgct gttactttaa gacttacaac
6601 agaagaaaat gagatattaa atagaatcaa agaaaaatat aatattagca aatcagatgc
6661 aaccggtatt ctaataaaaa aatatgcaaa ggaggaatac ggtgcatttt aaacaaaaaa
6721 agatagacag cactggcatg ctgcctatct atgactaaat tttgttaaat gtattagcac
6781 cgttattata tcatgagcga aaatgtaata aaagaaactg aaaacaagaa aaattcaaga
6841 ggacgtaatt ggacatttgt tttatatcca gaatcagcaa aagccgagtg gttagagtat
6901 ttaaaagagt tacacattca atttgtagtg tctccattac atgataggga tactgataca
6961 gaagatagga tgaaaaaaga gcattatcat attctagtga tgtatgaggg taataaatct
7021 tatgaacaga taaaaataat tacagaagaa ttgaatgcga ctattccgca gattgcagga
7081 agtgtgaaag gtcttgtgag atatatgctt cacatggacg atcctaataa atttaaatat
7141 caaaaagaag atatgatagt ttatggcggt gtagatgttg atgaattatt aaagaaaaca
7201 acaacagata gatataaatt aattaaagaa atgattgagt ttattgatga acaaggaatc
7261 gtagaattta agagtttaat ggattatgca atgaagttta aatttgatga ttggttcccg
7321 cttttatgtg ataactcggc gtatgttatt caagaatata taaaatcaaa tcggtataaa
7381 tctgaccgat agattttgaa tttaagagtg tcacaagaca ctcttttttc gcaccagcga
7441 aaactggttt aagccgactg cgcaaaagac ataatcgatt cacaaaaaat aggcacacga
7501 aaaacaagtt aagggatgca gtttatgcat cccttaactt acttattaaa taatttatag
7561 ctattgaaaa gagataagaa ttgttcaaag ctaatattgt ttaaatcgtc aattcctgca
7621 tgttttaagg aattgttaaa ttgatttttt gtaaatattt tcttgtattc tttgttatct
7681 tggttacccg tcttcttaat atgcgtaatt gcatctactc taaatccatc aatgccttta
7741 tcaaaccacc agttcatcat ttcaaataca gcatctctaa cttccggatt accccaattc
7801 aaatcaggtt gttttttact gaataaatgg aaataatatt gctcagtatt agcatcatat
7861 tcccgcgcgc caattgagct cccctttcta tgtatgtttt ttactagtca tttaaaacga
7921 tacattaata ggtacgaaaa agcaactttt tttgcgctta aaaccagtca taccaataac
7981 ttaagggtaa ctagcctcgc cggcaatagt tacccttatt atcaagataa gaaagaaaag
8041 gatttttcgc tacgctcaaa tcctttaaaa aaacacaaaa gaccacattt tttaatgtgg
8101 tcttttattc ttcaactaaa gcacccatta gttcaacaaa cgaaaattgg ataaagtggg
8161 atatttttaa aatatatatt tatgttacag taatattgac ttttaaaaaa ggattgattc
8221 taatgaagaa agcagacaag taagcctcct aaattcactt tagataaaaa tttaggaggc
8281 atatcaaatg aaatttaata aaattgattt agacaattgg aagagaaaag agatatttaa
8341 tcattatttg aaccaacaaa cgacttttag tataaccaca gaaattgata ttagtgtttt
8401 ataccgaaac ataaaacaag aaggatataa attttaccct gcatttattt tcttagtgac
8461 aagggtgata aactcaaata cagcttttag aactggttac aatagcgacg gagagttagg
8521 ttattgggat aagttagagc cactttatac aatttttgat ggtgtatcta aaacattctc
8581 tggtatttgg actcctgtaa agaatgactt caaagagttt tatgatttat acctttctga
8641 tgtagagaaa tataatggtt cggggaaatt gtttcccaaa acacctatac ctgaaaatgc
8701 tttttctctt tctattattc catggacttc atttactggg tttaacttaa atatcaataa
8761 taatagtaat taccttctac ccattattac agcaggaaaa ttcattaata aaggtaattc
8821 aatatattta ccgctatctt tacaggtaca tcattctgtt tgtgatggtt atcatgcagg
8881 attgtttatg aactctattc aggaattgtc agataggcct aatgactggc ttttataata
8941 tgagataatg ccgactgtac tttttacagt cggttttcta atgtcactaa cctgccccgt
9001 tagttgaaga aggtttttat attacagctc cagatccata tccttctttt tctgaaccga
9061 cttctccttt ttcgcttctt tattccaatt gctttattga cgttgagcct cggaaccggt
9121 acccaggaaa cagctatgac catgtaatac gactcactat acggggatat cgtcggaatt
9181 gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt
9241 cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga caggatgagg
9301 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
9361 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
9421 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
9481 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
9541 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
9601 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
9661 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
9721 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
9781 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
9841 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
9901 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
9961 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
10021 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
10081 tcgccttctt gacgagttct tctgagcggg actctggggt tcgagagctc gcttggactc
10141 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
10201 tgccgccggg cgttttttat tggtgagaat ccaagcact
//