LOCUS       sequence-181452-        7782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LwCas13a for bacterial expression and contains backbone 
            for spacer cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2017 Oct 4. doi: 10.1038/nature24049."
COMMENT     Sequence Label: pC019 - LwCas13a from Leptotrichia wadei
FEATURES             Location/Qualifiers
     source          1..7782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(55..60,61..78,79..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(join(7254..7782,1..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
//
