LOCUS Exported 7790 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION LwCas13a locus into pACYC184 with nontargeting spacer. ACCESSION . VERSION . KEYWORDS pC012 LwCas13a locus with nontargeting spacer SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7790) AUTHORS Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F TITLE Nucleic acid detection with CRISPR-Cas13a/C2c2. JOURNAL Science. 2017 Apr 13. pii: eaam9321. doi: 10.1126/science.aam9321. PUBMED 28408723 REFERENCE 2 (bases 1 to 7790) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7790 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(154..188) /label=J23119 promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119)" CDS complement(192..3647) /codon_start=1 /product="Leptotrichia wadei CRISPR-Cas effector that acts as an RNA-guided RNAse (Abudayyeh et al., 2017)" /label=LwCas13a /note="previously called C2c2" /translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE NENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE" promoter complement(3662..3691) /label=lac promoter /note="promoter for the E. coli lac operon" rep_origin 3729..4274 /direction=RIGHT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind 4175..4194 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4391..4408 /label=L4440 /note="L4440 vector, forward primer" promoter 4800..4902 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 4903..5562 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(4969..4988) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS complement(6596..7786) /codon_start=1 /gene="tet" /product="tetracycline efflux protein" /label=TcR /note="confers resistance to tetracycline" /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST" primer_bind 7415..7434 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag cttgttttag 61 tccccttcgt ttttggggta gtctaaatca gattgctgtt ctaccaagta atccatagtt 121 ttagtcccct tcgtttttgg ggtagtctaa atcgctagca ttatacctag gactgagcta 181 gctgtcaatc attccagggc cttgtactcg aacatcactt tcacgagttc gcacagttcc 241 tcgctgttcc ggtcggtcat cagtttcttt ttcttcagat tcttcaggtg cacgatcttc 301 tctgattcca gggtctggat ttcgatcttc ttgtcagcgc cgatcttgaa ggtggccacg 361 aagccgtatt ctttcagaat gtccacgatg gacttcatga tggcgttctt cagcttccgg 421 tcgtaggaca gcagcttccg caggttttcc agcacttcca gcaggctaat ctcggcgtgg 481 gggatgtagt tgaagtgggc aatgtagttc cggatgtaca ggtccttttt ttcctgcttc 541 agtttcttca ctttcttgtc ggagtagatg ctccgctttt ccacattgtc cttgtacagt 601 tctttgtaga agttgatata cttttccacg atctggccgc ttttgtactt cacattcttg 661 gagttgtcga aattgaaaat ttcctcgatg tagtggttct cgggaaactc gcccttcagc 721 cggaatctca ggtcccgctc ccagatgctg gtgtagccca cgagccggtg caggatcttc 781 agcagcaggc cctgcagcag gttcagctca ttgaattcca ccttgttctt caggtgggtg 841 tacttctgga tgttgccgat ggccttctca tactctttgt agtcctcgtc gttgaacttt 901 tcgtccttct tgggtctggc gtacttccgg tgcaggttct gctgcatggt gtagttcttt 961 tcaatctcat tcttcttgtt gctgtactct ttcagttctt tcaggctgat cttatacttg 1021 gccttatcgg cgatcttttc cagcagattc agcatgccgt atttcttgat attgtagaag 1081 gcccggtgct tgatgatgtt ctcgccgtcg aaatagatct tgttggtgtc gaactttttc 1141 agctctttcc ggtccttgat tttgttttcg ttgaagtcca ggaacttgcc gatctcgttg 1201 gcttccagct cgaagtcctc ggtcactctg ttgttgtcca ggttcagcag gttgatcagt 1261 tccagctcgt cgctgaaggt ttcttctttg ttggcggact ggtacttttc caggctgccc 1321 ttcaggttgg tcagctcttt gtggttcagc agcttcagga tcaggtaaaa catgttcaga 1381 ttctcggtgt acttcagaat cttccccagc ttgatctcgc gcacgaactc attgatctcg 1441 tgagggattt ctttgttccg attgtgcttc tcatagttct tcaggatctt gtcgtacttc 1501 tctttgttat cctttttgat cttgatcttg gagaagatgt cgttgttgtc attgttgttg 1561 ttgctctcga tatacttcag attgttcttg ttcaggtagt cgatgaagcc cttcaggaaa 1621 atctgctgaa taaagtcgat gtaggtattc ttttcctctt tgtcctggtt gttgatcatc 1681 tctctgctct ggatgatggc caggtattcc acgggcacgg ttttctcgat gttctcgaac 1741 ttctgatact tgtagtggcc ggttttctgg ttccgctgct tgttaatctt gatcacttca 1801 ttggtgatct taaagaacac cttggagttt ttcacgaact tgttcaggaa ctcgccgtag 1861 tagatattct tcagcaggta gatctgggcg tccttctctt ctttgtcctt gggcacgctc 1921 caaaaaaact tcagggtatt ccgcaggtcc tcaatcttgt tgtacagctt ggtgaagctg 1981 ggcacgaagg ggatgttttt gttcacgaag ttgaacttgg tattcttcag gtacttgatg 2041 atcacatcct tctcgtagta gttgaacacg ttggcgctgt tcagctgctt gaagattttc 2101 agcttcagct tcttttcgtt gatttcgttc tgaaacatct tcttggagat ctcgctgggg 2161 gcgatattct tgaaggcgaa gatgtccttg ccttccagtt ccaggttgaa gtgcacgatg 2221 ccgtgtctga tgctgctgat ggcctcgtcg atgttggcga agaagtcctc gatctcgttc 2281 ttgttgtcca tgttgaagtc gtagctgtag aacatcttca gattttcttt cacttcgttc 2341 tgcttgttct cattgtagat cttgtccacc tcgccggaca cgtatttctc ttcgcccttg 2401 ttgttcttca cggtcttgcc ccgcatccgg ccggtgatat cgttctcgtt ctcggtttcc 2461 aggatgttcc tcaggctgaa gtaggccacg ctggacacgc cgatgatgtt tctcaggaag 2521 gcctcgttct gccggttccg ggcgataaag tcggaggtgg cgatctcgcc cacttgcaga 2581 tagtagttgt acttgccgca gttccgcacg taggtgtcca gcttgttcag cagtttgttt 2641 tcgatcagct ttttcagatt ctggtactcg aagatccgct tgatcttatc gttgctgatg 2701 ttgctcagcc gcttgtacac gtagtttttc agcagctggg acatctcgat ttccacgaag 2761 tggcagaagg cgtacttaat attcttgtcg ttcagttcct ctttgtccag gtagtacttg 2821 tagaacacct ggcttttctt cagctcagac atgtcgggga tcttctcaat cagctctttg 2881 atgttgttca cgttctggat ctcttcgtag ataatcttgg cgaagttctc tttgtcgttc 2941 ttccggccga tgatcttgtg atagtactcg cggatcttgt acttctcgtg cttcttgctg 3001 ttctcgatca ggaaaaacag tttctcgata tcctctttct tatacagctt gtcgaaggct 3061 tcctgcacgt tgttgatgta gtcgttgcgc ttggcgctct ctctgtagta gtcgtagatg 3121 atgttccgct tgctcttgcc gcccactttt tccacgttgt tctcgttgat cttctggtag 3181 ttggccttgt tctcttcgaa gctgtacttc aggctgttga tcttgttcag cttggcttcc 3241 acgtccttcc gaaagatttc cagttcctca gagttcacgt cctcgttcag caggatcttc 3301 ttcagcacgg agaagctgtt cttgtttttc aggtcgtact cgctgatgtc ctcttcgcta 3361 tagttcttgt cctgcacggc gttcttttct ttccggttct tcagatacag cacgctgtcc 3421 ttcaggtgca gcaccttgtt gctaaagaac ttcttcaggt tctctcttct gatccggttc 3481 tcttcctcgg aggcgttgtc ggggttcttg atgtagatgt ccagccggat gctcagcagc 3541 tcgctcagtc tctcgctggt ccggttttcc tcgctggtgg acttcacgag cttgccctct 3601 tcgatgtact tcttgtggct gatgccgtcg accttggtca ctttcatgga taagacctcc 3661 taacatacga gccggaagca taaagtgtaa atctagaaat attttatctg attaataaga 3721 tgatcttctt gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc 3781 gccttgcagg gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac 3841 tggcttggag gagcgcagtc accaaaactt gtcctttcag tttagcctta accggcgcat 3901 gacttcaaga ctaactcctc taaatcaatt accagtggct gctgccagtg gtgcttttgc 3961 atgtctttcc gggttggact caagacgata gttaccggat aaggcgcagc ggtcggactg 4021 aacggggggt tcgtgcatac agtccagctt ggagcgaact gcctacccgg aactgagtgt 4081 caggcgtgga atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg 4141 caggaacagg agagcgcacg agggagccgc cagggggaaa cgcctggtat ctttatagtc 4201 ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc gtgatgcttg tcaggggggc 4261 ggagcctatg gaaaaacggc tttgccgcgg ccctctcact tccctgttaa gtatcttcct 4321 ggcatcttcc aggaaatctc cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac 4381 gaccgagcgt agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg 4441 ctgacgcacc ggtgcagcct tttttctcct gccacatgaa gcacttcact gacaccctca 4501 tcagtgccaa catagtaagc cagtatacac tccgctagcg ctgatgtccg gcggtgcttt 4561 tgccgttacg caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag 4621 ccactggagc acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc 4681 gacgcacttt gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat 4741 cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgacacgtt 4801 gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact accgggcgta 4861 ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact 4921 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag 4981 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag 5041 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg 5101 atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat 5161 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg 5221 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt 5281 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtgtca 5341 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc 5401 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg 5461 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat 5521 gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat 5581 tggtgccctt aaacgcctgg ttgctacgcc tgaataagtg ataataagcg gatgaatggc 5641 agaaattcga aagcaaattc gacccggtcg tcggttcagg gcagggtcgt taaatagccg 5701 cttatgtcta ttgctggttt accggtttat tgactaccgg aagcagtgtg accgtgtgct 5761 tctcaaatgc ctgaggccag tttgctcagg ctctccccgt ggaggtaata attgacgata 5821 tgatcattta ttctgcctcc cagagcctga taaaaacggt tagcgcttcg ttaatacaga 5881 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt 5941 gcagggcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg 6001 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac 6061 gatagtcatg ccccgcgccc accggaagga gctaccggca gcggtgcgga ctgttgtaac 6121 tcagaataag aaatgaggcc gctcatggcg ttgactctca gtcatagtat cgtggtatca 6181 ccggttggtt ccactctctg ttgcgggcaa cttcagcagc acgtagggga cttccgcgtt 6241 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac 6301 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca 6361 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc 6421 cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac 6481 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga 6541 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg 6601 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg 6661 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg 6721 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa 6781 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc 6841 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga 6901 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct 6961 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc 7021 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga 7081 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca 7141 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 7201 ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca 7261 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg 7321 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca 7381 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag 7441 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatccaca 7501 ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc gaagcgagca 7561 ggactgggcg gcggccaaag cggtcggaca gtgctccgag aacgggtgcg catagaaatt 7621 gcatcaacgc atatagcgct agcagcacgc catagtgact ggcgatgctg tcggaatgga 7681 cgatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca 7741 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac //