LOCUS Exported 7782 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses LwCas13a for bacterial expression and contains backbone for spacer cloning. ACCESSION . VERSION . KEYWORDS pC019 - LwCas13a from Leptotrichia wadei SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7782) AUTHORS Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F TITLE RNA targeting with CRISPR-Cas13. JOURNAL Nature. 2017 Oct 4. doi: 10.1038/nature24049. PUBMED 28976959 REFERENCE 2 (bases 1 to 7782) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7782 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 55..84 /label=lac promoter /note="promoter for the E. coli lac operon" CDS 99..3554 /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 3558..3592 /label=J23119 promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119)" CDS 3742..4932 /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 complement(4094..4113) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" CDS complement(5966..6625) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 6540..6559 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(6626..6728) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind complement(7120..7137) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7254..17) /direction=LEFT /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 complement(7334..7353) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc tagatttaca 61 ctttatgctt ccggctcgta tgttaggagg tcttatccat gaaagtgacc aaggtcgacg 121 gcatcagcca caagaagtac atcgaagagg gcaagctcgt gaagtccacc agcgaggaaa 181 accggaccag cgagagactg agcgagctgc tgagcatccg gctggacatc tacatcaaga 241 accccgacaa cgcctccgag gaagagaacc ggatcagaag agagaacctg aagaagttct 301 ttagcaacaa ggtgctgcac ctgaaggaca gcgtgctgta tctgaagaac cggaaagaaa 361 agaacgccgt gcaggacaag aactatagcg aagaggacat cagcgagtac gacctgaaaa 421 acaagaacag cttctccgtg ctgaagaaga tcctgctgaa cgaggacgtg aactctgagg 481 aactggaaat ctttcggaag gacgtggaag ccaagctgaa caagatcaac agcctgaagt 541 acagcttcga agagaacaag gccaactacc agaagatcaa cgagaacaac gtggaaaaag 601 tgggcggcaa gagcaagcgg aacatcatct acgactacta cagagagagc gccaagcgca 661 acgactacat caacaacgtg caggaagcct tcgacaagct gtataagaaa gaggatatcg 721 agaaactgtt tttcctgatc gagaacagca agaagcacga gaagtacaag atccgcgagt 781 actatcacaa gatcatcggc cggaagaacg acaaagagaa cttcgccaag attatctacg 841 aagagatcca gaacgtgaac aacatcaaag agctgattga gaagatcccc gacatgtctg 901 agctgaagaa aagccaggtg ttctacaagt actacctgga caaagaggaa ctgaacgaca 961 agaatattaa gtacgccttc tgccacttcg tggaaatcga gatgtcccag ctgctgaaaa 1021 actacgtgta caagcggctg agcaacatca gcaacgataa gatcaagcgg atcttcgagt 1081 accagaatct gaaaaagctg atcgaaaaca aactgctgaa caagctggac acctacgtgc 1141 ggaactgcgg caagtacaac tactatctgc aagtgggcga gatcgccacc tccgacttta 1201 tcgcccggaa ccggcagaac gaggccttcc tgagaaacat catcggcgtg tccagcgtgg 1261 cctacttcag cctgaggaac atcctggaaa ccgagaacga gaacgatatc accggccgga 1321 tgcggggcaa gaccgtgaag aacaacaagg gcgaagagaa atacgtgtcc ggcgaggtgg 1381 acaagatcta caatgagaac aagcagaacg aagtgaaaga aaatctgaag atgttctaca 1441 gctacgactt caacatggac aacaagaacg agatcgagga cttcttcgcc aacatcgacg 1501 aggccatcag cagcatcaga cacggcatcg tgcacttcaa cctggaactg gaaggcaagg 1561 acatcttcgc cttcaagaat atcgccccca gcgagatctc caagaagatg tttcagaacg 1621 aaatcaacga aaagaagctg aagctgaaaa tcttcaagca gctgaacagc gccaacgtgt 1681 tcaactacta cgagaaggat gtgatcatca agtacctgaa gaataccaag ttcaacttcg 1741 tgaacaaaaa catccccttc gtgcccagct tcaccaagct gtacaacaag attgaggacc 1801 tgcggaatac cctgaagttt ttttggagcg tgcccaagga caaagaagag aaggacgccc 1861 agatctacct gctgaagaat atctactacg gcgagttcct gaacaagttc gtgaaaaact 1921 ccaaggtgtt ctttaagatc accaatgaag tgatcaagat taacaagcag cggaaccaga 1981 aaaccggcca ctacaagtat cagaagttcg agaacatcga gaaaaccgtg cccgtggaat 2041 acctggccat catccagagc agagagatga tcaacaacca ggacaaagag gaaaagaata 2101 cctacatcga ctttattcag cagattttcc tgaagggctt catcgactac ctgaacaaga 2161 acaatctgaa gtatatcgag agcaacaaca acaatgacaa caacgacatc ttctccaaga 2221 tcaagatcaa aaaggataac aaagagaagt acgacaagat cctgaagaac tatgagaagc 2281 acaatcggaa caaagaaatc cctcacgaga tcaatgagtt cgtgcgcgag atcaagctgg 2341 ggaagattct gaagtacacc gagaatctga acatgtttta cctgatcctg aagctgctga 2401 accacaaaga gctgaccaac ctgaagggca gcctggaaaa gtaccagtcc gccaacaaag 2461 aagaaacctt cagcgacgag ctggaactga tcaacctgct gaacctggac aacaacagag 2521 tgaccgagga cttcgagctg gaagccaacg agatcggcaa gttcctggac ttcaacgaaa 2581 acaaaatcaa ggaccggaaa gagctgaaaa agttcgacac caacaagatc tatttcgacg 2641 gcgagaacat catcaagcac cgggccttct acaatatcaa gaaatacggc atgctgaatc 2701 tgctggaaaa gatcgccgat aaggccaagt ataagatcag cctgaaagaa ctgaaagagt 2761 acagcaacaa gaagaatgag attgaaaaga actacaccat gcagcagaac ctgcaccgga 2821 agtacgccag acccaagaag gacgaaaagt tcaacgacga ggactacaaa gagtatgaga 2881 aggccatcgg caacatccag aagtacaccc acctgaagaa caaggtggaa ttcaatgagc 2941 tgaacctgct gcagggcctg ctgctgaaga tcctgcaccg gctcgtgggc tacaccagca 3001 tctgggagcg ggacctgaga ttccggctga agggcgagtt tcccgagaac cactacatcg 3061 aggaaatttt caatttcgac aactccaaga atgtgaagta caaaagcggc cagatcgtgg 3121 aaaagtatat caacttctac aaagaactgt acaaggacaa tgtggaaaag cggagcatct 3181 actccgacaa gaaagtgaag aaactgaagc aggaaaaaaa ggacctgtac atccggaact 3241 acattgccca cttcaactac atcccccacg ccgagattag cctgctggaa gtgctggaaa 3301 acctgcggaa gctgctgtcc tacgaccgga agctgaagaa cgccatcatg aagtccatcg 3361 tggacattct gaaagaatac ggcttcgtgg ccaccttcaa gatcggcgct gacaagaaga 3421 tcgaaatcca gaccctggaa tcagagaaga tcgtgcacct gaagaatctg aagaaaaaga 3481 aactgatgac cgaccggaac agcgaggaac tgtgcgaact cgtgaaagtg atgttcgagt 3541 acaaggccct ggaatgattg acagctagct cagtcctagg tataatgcta gcgatttaga 3601 ctaccccaaa aacgaagggg actaaaacgg agacggagat tcgtctcgga tttagactac 3661 cccaaaaacg aaggggacta aaacaagctt taatgcggta gtttatcaca gttaaattgc 3721 taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc 3781 gtcaccctgg atgctgtagg cataggcttg gttatgccgg tactgccggg cctcttgcgg 3841 gatatcgtcc attccgacag catcgccagt cactatggcg tgctgctagc gctatatgcg 3901 ttgatgcaat ttctatgcgc acccgttctc ggagcactgt ccgaccgctt tggccgccgc 3961 ccagtcctgc tcgcttcgct acttggagcc actatcgact acgcgatcat ggcgaccaca 4021 cccgtcctgt ggatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt 4081 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc 4141 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg 4201 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac 4261 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg accgatgccc 4321 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc 4381 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg 4441 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg 4501 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt 4561 ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg ctacgtcttg 4621 ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct cgcttccggc 4681 ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga cgaccatcag 4741 ggacagcttc aaggatcgct cgcggctctt accagcctaa cttcgatcat tggaccgctg 4801 atcgtcacgg cgatttatgc cgcctcggcg agcacatgga acgggttggc atggattgta 4861 ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc 4921 acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaagaattg 4981 gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg cagaacatat 5041 ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc gttgggtcct 5101 ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt 5161 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct 5221 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa 5281 gtctggaaac gcggaagtcc cctacgtgct gctgaagttg cccgcaacag agagtggaac 5341 caaccggtga taccacgata ctatgactga gagtcaacgc catgagcggc ctcatttctt 5401 attctgagtt acaacagtcc gcaccgctgc cggtagctcc ttccggtggg cgcggggcat 5461 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc 5521 ggcagcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc 5581 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac 5641 acctacatct gtattaacga agcgctaacc gtttttatca ggctctggga ggcagaataa 5701 atgatcatat cgtcaattat tacctccacg gggagagcct gagcaaactg gcctcaggca 5761 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag 5821 acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg 5881 aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag 5941 ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt 6001 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa 6061 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg 6121 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg 6181 gattggctga cacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt 6241 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt 6301 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt 6361 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag 6421 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct 6481 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag 6541 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg 6601 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact 6661 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt 6721 gccgatcaac gtgtcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga 6781 caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa 6841 gtgcgtcggg tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct 6901 ccagtggctt ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt 6961 aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg 7021 gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt 7081 gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg 7141 ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga 7201 agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat 7261 aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac 7321 ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct 7381 gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc 7441 acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc 7501 ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga 7561 aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct 7621 tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc 7681 caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct 7741 gcaaggcggt tttttcgttt tcagagcaag agattacgcg ca //