LOCUS Exported 8761 bp ds-DNA circular SYN 11-OCT-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8761)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8761
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_RNA 225..300
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 434..777
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 778..1587
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
terminator 1593..1790
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
misc_feature 1855..2558
/label=RNR2 promoter
CDS 2559..6662
/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 6672..6692
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6708..6731
/codon_start=1
/product="8xHis affinity tag"
/label=8xHis
/translation="HHHHHHHH"
terminator 6735..6924
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
rep_origin complement(6987..7575)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind complement(8566..8613)
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
1 gtatatgtgt tatgtagtat actctttctt caacaattaa atactctcgg tagccaagtt
61 ggtttaaggc gcaagactgt aatttatcac tacgaaatct tgagatcggg cgttcgactc
121 gcccccggga gagatggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca
181 ccttcgggtg gcgaatggga ctttcataat aacgtccaat aaatgtttta gagctagaaa
241 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
301 tttttttatt ttttgtcact attgttatgt aaaatgccac ctctgacagt atggaacgca
361 aacttctgtc tagtggatat aggtctagag atctgtttag cttgcctcgt ccccgccggg
421 tcacccggcc agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag
481 ctcaggggca tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc
541 atttgcatcc atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct
601 gcagacctgc gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc
661 gcccctgtag agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact
721 tccttttaaa atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg
781 ggtaaggaaa agactcacgt ttcgaggccg cgattaaatt ccaacatgga tgctgattta
841 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
901 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
961 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
1021 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggc
1081 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
1141 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
1201 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
1261 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
1321 aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
1381 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
1441 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
1501 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
1561 catttgatgc tcgatgagtt tttctaatca gtactgacaa taaaaagatt cttgttttca
1621 agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta
1681 gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc
1741 agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc
1801 gatactaacg ccgccatcca gtgtcgaaaa cgagctctcg agaaccctta atgtcgacag
1861 tcgaacaaga agcaggcaaa gtttagagca ctgcccctcc gcactcaaaa aagaaaaaac
1921 taggaggaaa ataaaattct caaccacaca aacacataaa cacatacaaa tacaaataca
1981 agcttattta cttgacatcg cgcgatcttc cactattcag cgccgtccgc cctctctcgt
2041 gttttttgtt tacgcgacaa ctatgcgaaa tccggagcaa cgggcaaccg tttggggaaa
2101 gaccacaccc acgcgcgatc gccatggcaa cgaggtcgca cacgccccac acccagacct
2161 ccctgcgagc gggcatgggt acaatgtccc cgttgccaca gacaccactt cgtagcacag
2221 cgcagagcgt agcgtgttgt tgctgctgac aaaagaaaat ttttcttagc aaagcaaagg
2281 aggggaagca cgggcagata gcaccgtacc atacccttgg aaactcgaaa tgaacgaagc
2341 aggaaatgag agaatgagag ttttgtaggt atatatagcg gtagtgtttg cgcgttacca
2401 tcatcttctg gatctatcta ttgttctttt cctcatcact ttcccctttt tcgctcttct
2461 tcttgtcttt tatttctttc ttttttttaa ttgttccctc gattggctat ctaccaaaga
2521 atccaaactt aatacacgta tttatttgtc caattaccat ggataagaaa tactcaatag
2581 gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa tataaggttc
2641 cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa aaaaatctta
2701 taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc aaacggacag
2761 ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag attttttcaa
2821 atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct tttttggtgg
2881 aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat gaagttgctt
2941 atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat tctactgata
3001 aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt cgtggtcatt
3061 ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta tttatccagt
3121 tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt ggagtagatg
3181 ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat ctcattgctc
3241 agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg tcattgggtt
3301 tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta cagctttcaa
3361 aagatactta cgatgatgat ttagataatt tattggcgca aattggagat caatatgctg
3421 atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat atcctaagag
3481 taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc tacgatgaac
3541 atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca gaaaagtata
3601 aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat gggggagcta
3661 gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat ggtactgagg
3721 aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg
3781 gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga agacaagaag
3841 acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg acttttcgaa
3901 ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga
3961 agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa ggtgcttcag
4021 ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat gaaaaagtac
4081 taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg acaaaggtca
4141 aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag aagaaagcca
4201 ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta aaagaagatt
4261 atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa gatagattta
4321 atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa gattttttgg
4381 ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc ttatttgaag
4441 atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat gataaggtga
4501 tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga aaattgatta
4561 atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa tcagatggtt
4621 ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt aaagaagaca
4681 ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt gcaaatttag
4741 ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt gatgaattgg
4801 tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca cgtgaaaatc
4861 agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta
4921 tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact caattgcaaa
4981 atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg gaccaagaat
5041 tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa agtttcctta
5101 aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt ggtaaatcgg
5161 ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga caacttctaa
5221 acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt
5281 tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact cgccaaatca
5341 ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat gaaaatgata
5401 aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct gacttccgaa
5461 aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt
5521 atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt gaatcggagt
5581 ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag tctgagcaag
5641 aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac ttcttcaaaa
5701 cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa actaatgggg
5761 aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc aaagtattgt
5821 ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg
5881 agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa gactgggatc
5941 caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta gtggttgcta
6001 aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta gggatcacaa
6061 ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct aaaggatata
6121 aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt gagttagaaa
6181 acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat gagctggctc
6241 tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag ttgaagggta
6301 gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat tatttagatg
6361 agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat gccaatttag
6421 ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa caagcagaaa
6481 atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt aaatattttg
6541 atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat gccactctta
6601 tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag ctaggaggtg
6661 acggtggagg gccaaaaaag aaaagaaaag ttgaagatgc ttctgctcat catcaccatc
6721 accatcacca ttaaatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
6781 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
6841 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
6901 gagaaggttt tgggacgctc gaagccgcgg tgagcaaaag gccagcaaaa ggccaggaac
6961 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
7021 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
7081 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
7141 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
7201 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
7261 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
7321 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
7381 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
7441 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
7501 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
7561 aaaaaaggat ctcaagaaga tcctttgatc ttttctacac tagtcgaagc atctgtgctt
7621 cattttgtag aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
7681 tgcattttta cagaacagaa atgcaacgcg aaagcgctat tttaccaacg aagaatctgt
7741 gcttcatttt tgtaaaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc
7801 tgagctgcat ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa
7861 tctatacttc ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag
7921 catcttagat tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt
7981 ttgcactgta ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca
8041 taaaaaaagc ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat
8101 tttttcaaga taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt
8161 gtgaacagaa agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc
8221 ttctattttg tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga
8281 ttcactctat gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa
8341 acataaaaaa tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt
8401 aggttatata gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt
8461 gtggaagcgg tattcgcaat attttagtag ctcgttacag tccggtgcgt ttttggtttt
8521 ttgaaagtgc gtcttcagag cgcttttggt tttcaaaagc gctctgaagt tcctatactt
8581 tctagagaat aggaacttcg gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc
8641 gaaaatgcaa cgcgagctgc gcacatacag ctcactgttc acgtcgcacc tatatctgcg
8701 tgttgcctgt atatatatat acatgagaag aacggcatag tgcgtgttta tgcttaaatg
8761 c
//