LOCUS Exported 8472 bp ds-DNA circular SYN 26-JUN-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8472) AUTHORS sa TITLE Direct Submission JOURNAL Exported Jun 26, 2018 from SnapGene Viewer 4.1.9 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8472 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 409..1035 /codon_start=1 /label=catfish-AICDA /translation="MSKLDSVLLTQRKFIYHYKNVRWARGRNETYLCFVVKKRNSPDSL SFDFGHLRNRSGCHVELLFLSYLGVLCPGFLGSGVDGVRVAYAITWFCSWSPCSNCAHR LSRFMSQMPNLRLRIFVSRLYFCDEEDSQEREGLRCLQRAGVQVTVMTYKDFFYCWQTF VAQNQKAFKAWDDLHQNSIRLSRKLQRILQPSESEDLRDGFALLGL" CDS 1084..5184 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9n /note="generates RNA-guided single strand nicks in DNA due to the D10 mutation in the RuvC-like catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" misc_feature 5197..5445 /label=UGI CDS 5458..5478 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5487..5504 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 5533..5757 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5828..5844) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 5852..5868 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5876..5906) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5921..5942 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(6230..6818) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6989..7849) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7850..7954) /gene="bla" /label=AmpR promoter enhancer join(8224..8472,1..131) /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagcaagctg 421 gacagtgtgc tgctgactca gaggaagttt atttaccact ataagaatgt gcgctgggct 481 cgtgggagga acgagaccta cctctgtttt gtggtcaaga aacgcaacag tcccgactcg 541 ctctccttcg acttcggaca cctgcgcaat cgttctggct gccatgtgga gcttctcttc 601 ctgagctatc ttggggtact gtgcccaggt ttcttgggtt ccggtgtgga tggtgtcagg 661 gtggcttatg ccatcacctg gttctgttcc tggtcaccct gttcaaactg tgcccatcgc 721 ctttctcgct tcatgtctca gatgcccaac ctgcggctgc gcatcttcgt ctcgcgcctc 781 tacttctgtg acgaggagga cagtcaagag agagagggac tccgttgctt gcagagggca 841 ggtgtgcaag tgacagtcat gacctataaa gattttttct actgttggca aacctttgtg 901 gctcaaaatc agaaggcttt caaggcttgg gacgaccttc accagaactc tatccgactg 961 tctcggaaac tacagcgaat cctgcagcct agtgagtctg aagacctgag ggatggcttc 1021 gctctgctgg gccttagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1081 agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1141 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1201 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1261 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1321 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1381 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1441 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1501 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1561 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1621 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1681 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1741 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1801 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1861 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1921 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 1981 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2041 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2101 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2161 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2221 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2281 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2341 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2401 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2461 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2521 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2581 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2641 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2701 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2761 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2821 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2881 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 2941 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3001 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3061 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3121 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3181 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3241 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3301 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3361 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3421 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3481 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3541 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3601 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3661 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3721 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3781 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3841 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3901 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 3961 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4021 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4081 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4141 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4201 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4261 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4321 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4381 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4441 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4501 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4561 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4621 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4681 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4741 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4801 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4861 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4921 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 4981 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5041 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5101 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5161 gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5221 aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5281 gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5341 agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5401 ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5461 aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5521 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5581 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5641 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5701 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5761 ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5821 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5881 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 5941 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6001 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6061 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6121 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6181 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6241 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6301 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6361 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6421 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6481 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6541 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6601 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6661 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6721 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6781 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6841 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6901 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 6961 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7021 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7081 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7141 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7201 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7261 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7321 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7381 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7441 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7501 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7561 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7621 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7681 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7741 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7801 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7861 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7921 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 7981 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8041 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8101 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8161 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8221 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8281 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8341 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8401 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8461 atcaagtgta tc //