LOCUS Exported 8442 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 8442) 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..8442 /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..1005 /codon_start=1 /label=A3A /translation="MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTS VKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW GSAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWD TFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN" misc_feature 724..726 /label=C106S CDS 1054..5154 /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 5167..5415 /label=UGI CDS 5428..5448 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5457..5474 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 5503..5727 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5798..5814) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 5822..5838 /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(5846..5876) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5891..5912 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(6200..6788) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6959..7819) /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(7820..7924) /gene="bla" /label=AmpR promoter enhancer join(8194..8442,1..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 ggaagccagc 421 ccagcatccg ggcccagaca cttgatggat ccacacatat tcacttccaa ctttaacaat 481 ggcattggaa ggcataagac ctacctgtgc tacgaagtgg agcgcctgga caatggcacc 541 tcggtcaaga tggaccagca caggggcttt ctacacaacc aggctaagaa tcttctctgt 601 ggcttttacg gccgccatgc ggagctgcgc ttcttggacc tggttccttc tttgcagttg 661 gacccggccc agatctacag ggtcacttgg ttcatctcct ggagcccctg cttctcctgg 721 ggcagtgccg gggaagtgcg tgcgttcctt caggagaaca cacacgtgag actgcgtatc 781 ttcgctgccc gcatctatga ttacgacccc ctatataagg aggcactgca aatgctgcgg 841 gatgctgggg cccaagtctc catcatgacc tacgatgaat ttaagcactg ctgggacacc 901 tttgtggacc accagggatg tcccttccag ccctgggatg gactagatga gcacagccaa 961 gccctgagtg ggaggctgcg ggccattctc cagaatcagg gaaacagcgg cagcgagact 1021 cccgggacct cagagtccgc cacacccgaa agtgataaaa agtattctat tggtttagcc 1081 atcggcacta attccgttgg atgggctgtc ataaccgatg aatacaaagt accttcaaag 1141 aaatttaagg tgttggggaa cacagaccgt cattcgatta aaaagaatct tatcggtgcc 1201 ctcctattcg atagtggcga aacggcagag gcgactcgcc tgaaacgaac cgctcggaga 1261 aggtatacac gtcgcaagaa ccgaatatgt tacttacaag aaatttttag caatgagatg 1321 gccaaagttg acgattcttt ctttcaccgt ttggaagagt ccttccttgt cgaagaggac 1381 aagaaacatg aacggcaccc catctttgga aacatagtag atgaggtggc atatcatgaa 1441 aagtacccaa cgatttatca cctcagaaaa aagctagttg actcaactga taaagcggac 1501 ctgaggttaa tctacttggc tcttgcccat atgataaagt tccgtgggca ctttctcatt 1561 gagggtgatc taaatccgga caactcggat gtcgacaaac tgttcatcca gttagtacaa 1621 acctataatc agttgtttga agagaaccct ataaatgcaa gtggcgtgga tgcgaaggct 1681 attcttagcg cccgcctctc taaatcccga cggctagaaa acctgatcgc acaattaccc 1741 ggagagaaga aaaatgggtt gttcggtaac cttatagcgc tctcactagg cctgacacca 1801 aattttaagt cgaacttcga cttagctgaa gatgccaaat tgcagcttag taaggacacg 1861 tacgatgacg atctcgacaa tctactggca caaattggag atcagtatgc ggacttattt 1921 ttggctgcca aaaaccttag cgatgcaatc ctcctatctg acatactgag agttaatact 1981 gagattacca aggcgccgtt atccgcttca atgatcaaaa ggtacgatga acatcaccaa 2041 gacttgacac ttctcaaggc cctagtccgt cagcaactgc ctgagaaata taaggaaata 2101 ttctttgatc agtcgaaaaa cgggtacgca ggttatattg acggcggagc gagtcaagag 2161 gaattctaca agtttatcaa acccatatta gagaagatgg atgggacgga agagttgctt 2221 gtaaaactca atcgcgaaga tctactgcga aagcagcgga ctttcgacaa cggtagcatt 2281 ccacatcaaa tccacttagg cgaattgcat gctatactta gaaggcagga ggatttttat 2341 ccgttcctca aagacaatcg tgaaaagatt gagaaaatcc taacctttcg cataccttac 2401 tatgtgggac ccctggcccg agggaactct cggttcgcat ggatgacaag aaagtccgaa 2461 gaaacgatta ctccatggaa ttttgaggaa gttgtcgata aaggtgcgtc agctcaatcg 2521 ttcatcgaga ggatgaccaa ctttgacaag aatttaccga acgaaaaagt attgcctaag 2581 cacagtttac tttacgagta tttcacagtg tacaatgaac tcacgaaagt taagtatgtc 2641 actgagggca tgcgtaaacc cgcctttcta agcggagaac agaagaaagc aatagtagat 2701 ctgttattca agaccaaccg caaagtgaca gttaagcaat tgaaagagga ctactttaag 2761 aaaattgaat gcttcgattc tgtcgagatc tccggggtag aagatcgatt taatgcgtca 2821 cttggtacgt atcatgacct cctaaagata attaaagata aggacttcct ggataacgaa 2881 gagaatgaag atatcttaga agatatagtg ttgactctta ccctctttga agatcgggaa 2941 atgattgagg aaagactaaa aacatacgct cacctgttcg acgataaggt tatgaaacag 3001 ttaaagaggc gtcgctatac gggctgggga cgattgtcgc ggaaacttat caacgggata 3061 agagacaagc aaagtggtaa aactattctc gattttctaa agagcgacgg cttcgccaat 3121 aggaacttta tgcagctgat ccatgatgac tctttaacct tcaaagagga tatacaaaag 3181 gcacaggttt ccggacaagg ggactcattg cacgaacata ttgcgaatct tgctggttcg 3241 ccagccatca aaaagggcat actccagaca gtcaaagtag tggatgagct agttaaggtc 3301 atgggacgtc acaaaccgga aaacattgta atcgagatgg cacgcgaaaa tcaaacgact 3361 cagaaggggc aaaaaaacag tcgagagcgg atgaagagaa tagaagaggg tattaaagaa 3421 ctgggcagcc agatcttaaa ggagcatcct gtggaaaata cccaattgca gaacgagaaa 3481 ctttacctct attacctaca aaatggaagg gacatgtatg ttgatcagga actggacata 3541 aaccgtttat ctgattacga cgtcgatcac attgtacccc aatccttttt gaaggacgat 3601 tcaatcgaca ataaagtgct tacacgctcg gataagaacc gagggaaaag tgacaatgtt 3661 ccaagcgagg aagtcgtaaa gaaaatgaag aactattggc ggcagctcct aaatgcgaaa 3721 ctgataacgc aaagaaagtt cgataactta actaaagctg agaggggtgg cttgtctgaa 3781 cttgacaagg ccggatttat taaacgtcag ctcgtggaaa cccgccaaat cacaaagcat 3841 gttgcacaga tactagattc ccgaatgaat acgaaatacg acgagaacga taagctgatt 3901 cgggaagtca aagtaatcac tttaaagtca aaattggtgt cggacttcag aaaggatttt 3961 caattctata aagttaggga gataaataac taccaccatg cgcacgacgc ttatcttaat 4021 gccgtcgtag ggaccgcact cattaagaaa tacccgaagc tagaaagtga gtttgtgtat 4081 ggtgattaca aagtttatga cgtccgtaag atgatcgcga aaagcgaaca ggagataggc 4141 aaggctacag ccaaatactt cttttattct aacattatga atttctttaa gacggaaatc 4201 actctggcaa acggagagat acgcaaacga cctttaattg aaaccaatgg ggagacaggt 4261 gaaatcgtat gggataaggg ccgggacttc gcgacggtga gaaaagtttt gtccatgccc 4321 caagtcaaca tagtaaagaa aactgaggtg cagaccggag ggttttcaaa ggaatcgatt 4381 cttccaaaaa ggaatagtga taagctcatc gctcgtaaaa aggactggga cccgaaaaag 4441 tacggtggct tcgatagccc tacagttgcc tattctgtcc tagtagtggc aaaagttgag 4501 aagggaaaat ccaagaaact gaagtcagtc aaagaattat tggggataac gattatggag 4561 cgctcgtctt ttgaaaagaa ccccatcgac ttccttgagg cgaaaggtta caaggaagta 4621 aaaaaggatc tcataattaa actaccaaag tatagtctgt ttgagttaga aaatggccga 4681 aaacggatgt tggctagcgc cggagagctt caaaagggga acgaactcgc actaccgtct 4741 aaatacgtga atttcctgta tttagcgtcc cattacgaga agttgaaagg ttcacctgaa 4801 gataacgaac agaagcaact ttttgttgag cagcacaaac attatctcga cgaaatcata 4861 gagcaaattt cggaattcag taagagagtc atcctagctg atgccaatct ggacaaagta 4921 ttaagcgcat acaacaagca cagggataaa cccatacgtg agcaggcgga aaatattatc 4981 catttgttta ctcttaccaa cctcggcgct ccagccgcat tcaagtattt tgacacaacg 5041 atagatcgca aacgatacac ttctaccaag gaggtgctag acgcgacact gattcaccaa 5101 tccatcacgg gattatatga aactcggata gatttgtcac agcttggggg tgactctggt 5161 ggttctacta atctgtcaga tattattgaa aaggagaccg gtaagcaact ggttatccag 5221 gaatccatcc tcatgctccc agaggaggtg gaagaagtca ttgggaacaa gccggaaagc 5281 gatatactcg tgcacaccgc ctacgacgag agcaccgacg agaatgtcat gcttctgact 5341 agcgacgccc ctgaatacaa gccttgggct ctggtcatac aggatagcaa cggtgagaac 5401 aagattaaga tgctctctgg tggttctccc aagaagaaga ggaaagtcta accggtcatc 5461 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc 5521 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 5581 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 5641 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 5701 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga 5761 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 5821 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 5881 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 5941 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 6001 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 6061 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 6121 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6181 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 6241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6361 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 6421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 6481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 6541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 6601 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 6661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 6721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 6781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 6841 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 6901 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 6961 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 7021 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 7081 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 7141 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 7201 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 7261 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 7321 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 7381 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 7441 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 7501 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 7561 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 7621 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 7681 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 7741 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 7801 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 7861 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 7921 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg 7981 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc 8041 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa 8101 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc 8161 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat 8221 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 8281 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 8341 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 8401 atttacggta aactgcccac ttggcagtac atcaagtgta tc //