LOCUS Exported 8783 bp ds-DNA circular SYN 28-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pRH2502
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8783)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 28, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8783
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 1..4104
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4117..4137
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 4158..4292
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(4488..5076)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5407..6222)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
CDS complement(6522..7520)
/codon_start=1
/label=Int
/translation="MDAEAWLAGEKRLIEMETWTPPQDRAKKAAASAITLEEYTRKWLV
ERDLADGTRDLYSGHAERRIYPVLGEVAVTEMTPALVRAWWAGMGRKHPTARRHAYNVL
RAVMNTAVEDKLIAENPCRIEQKAADERDVEALTPEELDIVAAEIFVHYRIAAYILAWT
SLRFGELIELRRKDIVDDGMTMKLRVRRGASRVGNKIVVGNAKTVRSKRPVTVPPHVAE
MIRAHMKDRTKMNKGPEAFLVTTTQGNRLSKSAFTKSLKRGYAKIGRPELRIHDLRAVG
ATFAAQAGATTKELMARLGHTTPRMAMKYQMASEARDEAIAEAMSKLAKTS"
terminator 8170..8216
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
misc_feature 8449..8625
/label=uvtetO promoter
protein_bind 8626..8643
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
1 atggacaaga agtacagcat cggcctggcg atcggcacga actcggtggg ctgggcggtc
61 atcacggacg agtacaaggt cccctccaag aagttcaagg tcctgggcaa caccgaccgg
121 cacagcatca agaagaacct gatcggcgcc ctgctgttcg actccggcga gaccgccgag
181 gcgacgcgcc tgaagcggac cgcgcggcgc cgatacacgc ggcgcaagaa ccgcatctgc
241 tacctgcagg aaatcttctc gaacgagatg gccaaggtgg acgacagctt cttccaccgg
301 ctggaggagt ccttcctggt ggaggaggac aagaagcacg agcgccaccc gatcttcggc
361 aacatcgtgg acgaggtggc ctaccacgag aagtacccca cgatctacca cctgcgcaag
421 aagctggtgg acagcaccga caaggcggac ctgcggctga tctacctggc cctggcgcac
481 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaacccgga caactcggac
541 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc
601 atcaacgcct ccggcgtgga cgccaaggcg atcctgtcgg cgcgcctgtc caagtcgcgg
661 cgcctggaga acctgatcgc ccagctgccg ggcgagaaga agaacggcct gttcggcaac
721 ctgatcgcgc tgagcctggg cctgacgccc aacttcaagt ccaacttcga cctggccgag
781 gacgcgaagc tgcagctgtc gaaggacacc tacgacgacg acctggacaa cctgctggcc
841 cagatcggcg accagtacgc ggacctgttc ctggccgcca agaacctgag cgacgccatc
901 ctgctgtccg acatcctgcg ggtcaacacc gagatcacga aggccccgct gagcgcgtcc
961 atgatcaagc ggtacgacga gcaccaccag gacctgaccc tgctgaaggc gctggtgcgc
1021 cagcagctgc ccgagaagta caaggaaatc ttcttcgacc agtcgaagaa cggctacgcc
1081 ggctacatcg acggcggcgc gagccaggag gagttctaca agttcatcaa gccgatcctg
1141 gagaagatgg acggcacgga ggagctgctg gtcaagctga accgcgagga cctgctgcgc
1201 aagcagcgga ccttcgacaa cggctcgatc ccgcaccaga tccacctggg cgagctgcac
1261 gccatcctgc ggcgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc
1321 gagaagatcc tgaccttccg catcccgtac tacgtgggcc ccctggcccg cggcaactcg
1381 cggttcgcgt ggatgacccg gaagagcgag gagaccatca cgccgtggaa cttcgaggag
1441 gtcgtggaca agggcgcctc ggcgcagagc ttcatcgagc gcatgaccaa cttcgacaag
1501 aacctgccga acgagaaggt cctgcccaag cacagcctgc tgtacgagta cttcaccgtg
1561 tacaacgagc tgacgaaggt caagtacgtg accgagggca tgcggaagcc ggccttcctg
1621 tcgggcgagc agaagaaggc gatcgtggac ctgctgttca agaccaaccg caaggtcacg
1681 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gtttcgactc ggtcgagatc
1741 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc
1801 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtc
1861 ctgaccctga cgctgttcga ggaccgcgag atgatcgagg agcggctgaa gacgtacgcc
1921 cacctgttcg acgacaaggt gatgaagcag ctgaagcggc gccgatacac cggctggggc
1981 cgcctgtcgc ggaagctgat caacggcatc cgggacaagc agtccggcaa gaccatcctg
2041 gacttcctga agtcggacgg cttcgcgaac cgcaacttca tgcagctgat ccacgacgac
2101 agcctgacct tcaaggagga catccagaag gcccaagtga gcggccaggg cgactccctg
2161 cacgagcaca tcgccaacct ggcgggctcg ccggcgatca agaagggcat cctgcagacc
2221 gtcaaggtcg tggacgagct ggtcaaggtg atgggccgcc acaagcccga gaacatcgtg
2281 atcgagatgg cccgggagaa ccagaccacg cagaagggcc agaagaacag ccgcgagcgg
2341 atgaagcgca tcgaggaggg catcaaggag ctgggctcgc agatcctgaa ggagcacccg
2401 gtcgagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc
2461 gacatgtacg tggaccagga gctggacatc aaccggctga gcgactacga cgtggacgcc
2521 atcgtgccgc agtcgttcct gaaggacgac agcatcgaca acaaggtcct gacccgctcg
2581 gacaagaacc ggggcaagtc ggacaacgtg cccagcgagg aggtcgtgaa gaagatgaag
2641 aactactggc gccagctgct gaacgccaag ctgatcacgc agcgcaagtt cgacaacctg
2701 accaaggccg agcggggcgg cctgagcgag ctggacaagg cgggcttcat caagcgccag
2761 ctggtcgaga cccggcagat cacgaagcac gtggcgcaga tcctggactc gcggatgaac
2821 acgaagtacg acgagaacga caagctgatc cgcgaggtca aggtgatcac cctgaagtcc
2881 aagctggtct cggacttccg caaggacttc cagttctaca aggtccggga gatcaacaac
2941 taccaccacg cccacgacgc gtacctgaac gccgtcgtgg gcaccgcgct gatcaagaag
3001 tacccgaagc tggagtcgga gttcgtctac ggcgactaca aggtctacga cgtgcgcaag
3061 atgatcgcca agagcgagca ggagatcggc aaggccaccg cgaagtactt cttctactcc
3121 aacatcatga acttcttcaa gaccgagatc acgctggcca acggcgagat ccgcaagcgc
3181 ccgctgatcg agaccaacgg cgagacgggc gagatcgtct gggacaaggg ccgcgacttc
3241 gcgaccgtcc ggaaggtgct gagcatgccg caggtcaaca tcgtgaagaa gaccgaggtg
3301 cagacgggcg gcttctccaa ggagtcgatc ctgcccaagc gcaactcgga caagctgatc
3361 gcccggaaga aggactggga cccgaagaag tacggcggct tcgactcccc caccgtcgcc
3421 tactcggtgc tggtcgtggc gaaggtcgag aagggcaagt ccaagaagct gaagtcggtg
3481 aaggagctgc tgggcatcac catcatggag cgctcgtcgt tcgagaagaa cccgatcgac
3541 ttcctggagg cgaagggcta caaggaggtc aagaaggacc tgatcatcaa gctgcccaag
3601 tacagcctgt tcgagctgga gaacggccgc aagcggatgc tggcctccgc gggcgagctg
3661 cagaagggca acgagctggc cctgccgtcg aagtacgtca acttcctgta cctggcgtcg
3721 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag
3781 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagcgggtc
3841 atcctggccg acgcgaacct ggacaaggtg ctgtcggcct acaacaagca ccgcgacaag
3901 ccgatccggg agcaggcgga gaacatcatc cacctgttca ccctgacgaa cctgggcgcc
3961 cccgccgcgt tcaagtactt cgacaccacg atcgaccgca agcggtacac ctccacgaag
4021 gaggtgctgg acgcgaccct gatccaccag tcgatcaccg gcctgtacga gacgcgcatc
4081 gacctgagcc agctgggcgg cgactcccgg gcggacccga agaagaagcg gaaggtgtga
4141 ttaattaaca aactcggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
4201 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
4261 aactcatcaa tgtatcttat catgtctgga tcctctagag tcgacctgca ggtttaaacg
4321 cggccgcacg cgttgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
4381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
4441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
4501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
4561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
4621 accctgccgc ttaccggata cctgtccgcc tttctcccct cgggaagcgt ggcgctttct
4681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
4741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
4801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
4861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
4921 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
4981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
5041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
5101 gggtctgacg ctcatggaac tagtgaggtc tgcctcgtga agaaggtgtt gctgactcat
5161 accaggcctg aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttggtgagag
5221 ctttgttgta ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg
5281 cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac
5341 aaagccgccg tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa
5401 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
5461 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
5521 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
5581 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
5641 gacgactgaa tccggtgaga atggcaaaag attatgcatt tctttccaga cttgttcaac
5701 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
5761 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
5821 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
5881 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
5941 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
6001 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
6061 cagaaacaac tctggcgcat ggggcttccc atacaagcga tagattgtcg cacctgattg
6121 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
6181 tcgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
6241 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
6301 acatcagaga ttttgagaca caacgtggct ttcccccccc cccgacgtca ggtggctagc
6361 tggcgggaac ggcaatgctc ctggcggcgg cggggctggt gcccaggtct ccgcacagag
6421 cggcggtgct ggcgctcgcg gccaggcgtg gttcttcgcg tactgacaag aaacccccct
6481 ctttaggact cagtgtcctt gggagggggg ctttttgcgt ttcaggaggt cttggccagc
6541 ttggacatcg cctcagcgat agcctcgtcg cgggcctcag acgccatctg gtacttcatc
6601 gccatcctag gagtcgtgtg accgagacgg gccatcagct ccttggtcgt cgcacctgcc
6661 tgagcggcga acgtagcgcc gacagcgcgg aggtcgtgga tgcggagttc cggccgaccg
6721 atcttggcgt agccacgctt cagcgacttg gtgaacgcgg acttcgacag ccggttgccc
6781 tgcgtcgtgg tcaccaggaa tgcctcgggg cccttgttca tcttcgtacg gtccttcatg
6841 tgcgctcgga tcatctccgc gacgtgaggc ggaaccgtca caggacgctt cgaccggacg
6901 gtcttggcgt tgccaacgac gatcttgttc cccacgcggg aagcgccacg gcgcacccgg
6961 agcttcatcg tcatgccgtc gtccacgatg tccttgcggc gaagctcgat cagctctccg
7021 aaccggaggc tcgtccacgc caggatgtat gccgcgatcc ggtagtgcac gaagatctca
7081 gcggcgacga tgtccagctc ctcaggcgtc agcgcctcta cgtcgcgctc atcggctgcc
7141 ttctgctcga tccggcacgg gttctctgcg atcagcttgt cctcgaccgc tgtgttcatc
7201 accgcccgga ggacgttgta ggcatgccgg cgggcagtcg ggtgcttcct acccatcccg
7261 gcccaccacg cacgcaccag agctggcgtc atctctgtga ccgccacttc acctagcacc
7321 gggtagatgc ggcgctccgc gtgcccgctg tacagatccc tggtgccgtc tgcgaggtcg
7381 cgctccacga gccacttccg ggtgtactcc tccagcgtga tggcgctggc ggctgccttc
7441 ttcgcccggt cctgtggagg ggtccaggtc tccatctcga tgagccgctt ctcgcccgcg
7501 agccaggctt cggcgtccat cttgttgtcg taggtctgca gcgcgtagta cctcacaccg
7561 tcctgcgggt tgacgtatga ggcttggatc ctcccgctgc gctgagtctt cagcgatccc
7621 catccgcgac gtgccaacta ggtctcctct cgtcgtgaac aaggctaccg ggttgcaact
7681 cctgtgcaac tctcaggctt caacgcgctt ctacgacctg caatttcttt ccacttagag
7741 gatgcagccg agagggggta aaaacctatc ttgaccggcc catatatgtg gtcggcagac
7801 acccattctt ccaaactagc tacgcgggtt cgattcccgt cgcccgctcc gctggtcaga
7861 gggtgttttc gccctctggc catttttctt tccaggggtc tgcaactctt gtgcgactct
7921 tctgacctgg gcatacgcgg ttgcaacgca tccctgatct ggctactttc gatgctgaca
7981 aacgaataga gccccccgcc tgcgcgaaca gacgaggggc attcacacca gattggagct
8041 ggtgcagtga agagaataga ccgggacaag gttgcaccgg gagttgcagc ggtcggaacc
8101 ctcgccgtcg gcgggctggc gttcgccctg tcgttcacgg ctctagctga tcaccgcggc
8161 catgatggca taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
8221 ctggcagtcg atcgtacgct agttaactac gtcgacatcg atttaatcga tcgatatgca
8281 tcaattgatt taaatactag tgttaactat ttaattgggg accctagagg tccccttttt
8341 tattttaaaa attttttcaa ggggacctct agggtcccca gctggctagc gagtcatgag
8401 gtcaccatgg tggccatcta gacatcatcg gtaccaggcc tagatctact agaaatattg
8461 gatcgtcggc accgtcacgg ccgtgggagg cggcacgatc cgcgacgtga tgatcggccg
8521 catccccacg gtgctgcgca gtgagctcta cgccatcccg gcgttgatct gtgcgttcgc
8581 acgcacaggc ccggtgtgag aagggtctct gacgagcggg agaactccct atcagtgata
8641 gagtttgtcc tccctatcag tgatagatag gctctgggag tacccgtgtg tacgaccagc
8701 acggcataca tcatttcgac gccgagagat tcgccgcccg aaatgagcac gatccgctcg
8761 aggcatgcga aggagatata cat
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