LOCUS       sequence-181453-        7782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lw2Cas13a 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: pC029 - Lw2Cas13a from Leptotrichia wadei F0279
FEATURES             Location/Qualifiers
     source          1..7782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     420..439
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(941..975)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             complement(979..4434)
                     /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(join(4449..4454,4455..4472,4473..4478))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     rep_origin      4516..5061
                     /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     4962..4981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5178..5195
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5587..5689
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             5690..6349
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5756..5775)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(join(7383..7782,1..791))
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
ORIGIN
        1 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
       61 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
      121 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      181 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc tcccttatgc
      241 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
      301 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
      361 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
      421 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      481 atgcgtccgg cgtagaggat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata
      541 gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct
      601 ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag
      661 tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata
      721 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
      781 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
      841 taaagcttgt tttagatccc ttcgtttttg gggttatcta tatccgagac gaatctccgt
      901 ctccgtttta gatcccttcg tttttggggt tatctatatc gctagcatta tacctaggac
      961 tgagctagct gtcaatcact ccagggcctt gtactcgaac atcactttca ccagctcgca
     1021 cagctcctcg ctgttccgat ctgtcatcag cttcttcttc ttcaggttct tcaggtgcac
     1081 gatcttctcg ctctccaggg tctggatctc gatcttcttg tcggcgccga tcttgaaggt
     1141 ggccacgaag ccatactcct tcaggatatc cacgatgctc ttcatgatgg cattcttcag
     1201 cttgcggtcg taagacagca gcttcctcag gttctccagc acctccagca ggctgatctc
     1261 ggcgtgaggg atataattaa agtgggcgat gtagtttctg atatacaggt ccttcttctc
     1321 ctgcttcagc ttcttcacct tcttatcgga gtagatgctc cgcttctcca cattgtcctt
     1381 atacagctcc ttatagaagt tgatgtactt ctccacgatc tggccagact tgtacttcac
     1441 attcttggag ttatcgaaat tgaagatctc ctcgatgtag tggttctctg gaaactcgcc
     1501 cttcaggcgg aacctcaggt cgcgctccca gatggatgta tagcccacca gcctgtgcag
     1561 gatcttcagc agcaggccct gcagcaggtt cagctcatta aactccacct tattcttcag
     1621 gtgggtgtac ttctggatgt tgccgatggc cttctcatac tccttgtaat cctcgtcgtt
     1681 gaacttctca tccttcttag ggcgggcgta cttcctgtgc agattctgct gcatggtata
     1741 gttcttctcg atctcgttct tcttattgga gtactccttc agctccttca ggctgatctt
     1801 atacttggcc ttgtcggcga tcttctccag cagattcagc atgccgtact tcttgatgtt
     1861 atagaaggcg cggtgcttga tgatattctc gccatcaaag tagatcttgt ttgtgtcgaa
     1921 cttcttcagc tccttcctat ccttgatctt attctcgtta aagtccagga acttgccgat
     1981 ctcgttggcc tccagctcaa aatcctcggt cacccgattg ttgtccaggt tcagcagatt
     2041 gatcagctcc agctcatcgc tgaatgtctc ctccttgttg gcgctctggt acttctccag
     2101 agagcccttc agattggtca gctccttgtg gttcagcagc ttcaggatca ggtaaaacat
     2161 attcaggttc tctgtatact tcaggatctt gcccagcttg atctcgcgca cgaactcgtt
     2221 gatctcgtgg gggatctcct tattcctgtt gtgcttctcg taattcttca ggatcttatc
     2281 atacttctcc ttgttgtcct tcttgatctt gatcttagaa aagatatcgt tgttgtcgtt
     2341 gttgttgttg gactcgatgt acttcagatt gttcttattc agatagtcga taaagccctt
     2401 caggaagatc tgctggatga aatcgatata ggtgttcttc tcctccttgt cctgattgtt
     2461 gatcatctct ctgctctgga tgatggccag gtactccacg ggcacggttt tctcgatgtt
     2521 ctcgaacttc tgatacttgt agtggccggt tttctgattc cgctgcttgt tgatcttgat
     2581 cacttcattt gtgatcttaa agaacacctt gctgttcttc acaaacttgt tcaggaactc
     2641 gccatagtag atgttcttca gcaggtagat ctgggcatcc ttctcctcct tgtcctttgg
     2701 cacagaccaa aagaacttca gggtatttct cagatcctcg atcttgttat acagctttgt
     2761 aaaggaaggc acgaagggga tattcttgtt cacaaaattg aacttggtgt tcttcaggta
     2821 cttgatgatc acgtccttct catagtagtt gaacacgttg gcggagttca gctgcttgaa
     2881 gatcttcagc ttcagcttct tctcattgat ctcgttctga aacatcttct tagagatctc
     2941 ggatggggcg atattcttaa aggcgaagat gtccttgccc tccagctcca ggttgaagtg
     3001 cacgatgccg tgccggatgc tagagatggc ctcatcgata ttggcaaaga agtcctcgat
     3061 ctcattcttg ttatccatgt taaagtcata gctgtagaac atcttcagat tctccttcac
     3121 ctcgttctgc ttattctcgt tgtagatctt atccacctcg ccgctcacgt acttctcctc
     3181 gcccttattg ttcttcacgg tcttgcctct catccggcct gtgatgtcat tctcgttctc
     3241 tgtctccagg atatttctca gagaaaagta ggccacggag ctcacgccga tgatgttccg
     3301 cagaaaggcc tcattctggc ggttcctggc gatgaaatcg gatgtggcga tctcgcccac
     3361 ttgcagatag taattgtact tgccacagtt gcgcacatag gtgtccagct tattcagcag
     3421 cttgttctcg atcagcttct tcagattctg gtactcgaag attctcttga tcttgtcatt
     3481 gctgatgtta gacagccgct tatacacgta gttcttcagc agctggctca tctcgatctc
     3541 cacaaagtgg cagaaggcat acttgatatt cttatcgttc agctcctcct tgtccagata
     3601 gtacttgtaa aacacctggg acttcttcag ctcgctcata tcagggatct tctcgatcag
     3661 ctccttgata ttgttcacgt tctggatctc ctcatagatg atcttggcga aattctcctt
     3721 gtcgttcttt ctgccgatga tcttgtgata gtactcccgg atcttgtact tctcgtgctt
     3781 cttggagttc tcgatcagaa agaacagctt ctcgatatcc tccttcttat acagcttgtc
     3841 gaaggcctcc tgcacattgt tgatgtaatc atttctcttg gcggactccc gatagtagtc
     3901 atagatgatg ttgcgcttgc tcttgccgcc cactttctcc acattgttct cgttgatctt
     3961 ctggtaattg gccttgttct cctcaaagct atacttcaga gaattgatct tgttcagctt
     4021 ggcctccaca tccttcctga agatctccag ctcctcgcta ttcacgtcct cgttcagcag
     4081 gatcttcttc agcacggaga agctattctt gttcttcaga tcgtactcag agatgtcctc
     4141 ctcggaataa ttcttatcct gcacggcatt cttctccttc ctgttcttca ggtacagcac
     4201 gctgtccttc aggtgcagca ccttgttaga aaagaacttc ttcagattct ctctccggat
     4261 gcggttctcc tcctcggagg cattatcggg gttcttgatg tagatgtcca gcctgatgga
     4321 cagcagctcg ctcagtctct cagatgtccg gttctcctca gaggtggact tcaccagctt
     4381 gccctcctcg atgtacttct tgtggctgat gccgtccacc ttggtcacct tcatggataa
     4441 gacctcctaa catacgagcc ggaagcataa agtgtaaatc tagaaatatt ttatctgatt
     4501 aataagatga tcttcttgag atcgttttgg tctgcgcgta atctcttgct ctgaaaacga
     4561 aaaaaccgcc ttgcagggcg gtttttcgaa ggttctctga gctaccaact ctttgaaccg
     4621 aggtaactgg cttggaggag cgcagtcacc aaaacttgtc ctttcagttt agccttaacc
     4681 ggcgcatgac ttcaagacta actcctctaa atcaattacc agtggctgct gccagtggtg
     4741 cttttgcatg tctttccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4801 cggactgaac ggggggttcg tgcatacagt ccagcttgga gcgaactgcc tacccggaac
     4861 tgagtgtcag gcgtggaatg agacaaacgc ggccataaca gcggaatgac accggtaaac
     4921 cgaaaggcag gaacaggaga gcgcacgagg gagccgccag ggggaaacgc ctggtatctt
     4981 tatagtcctg tcgggtttcg ccaccactga tttgagcgtc agatttcgtg atgcttgtca
     5041 ggggggcgga gcctatggaa aaacggcttt gccgcggccc tctcacttcc ctgttaagta
     5101 tcttcctggc atcttccagg aaatctccgc cccgttcgta agccatttcc gctcgccgca
     5161 gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca
     5221 tattctgctg acgcaccggt gcagcctttt ttctcctgcc acatgaagca cttcactgac
     5281 accctcatca gtgccaacat agtaagccag tatacactcc gctagcgctg atgtccggcg
     5341 gtgcttttgc cgttacgcac caccccgtca gtagctgaac aggagggaca gctgatagaa
     5401 acagaagcca ctggagcacc tcaaaaacac catcatacac taaatcagta agttggcagc
     5461 atcacccgac gcactttgcg ccgaataaat acctgtgacg gaagatcact tcgcagaata
     5521 aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt ggcgaaaatg
     5581 acacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc
     5641 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     5701 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     5761 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     5821 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     5881 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     5941 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     6001 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     6061 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     6121 cgtgtcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     6181 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     6241 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     6301 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaagg
     6361 cagttattgg tgcccttaaa cgcctggttg ctacgcctga ataagtgata ataagcggat
     6421 gaatggcaga aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa
     6481 atagccgctt atgtctattg ctggtttacc ggtttattga ctaccggaag cagtgtgacc
     6541 gtgtgcttct caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt
     6601 gacgatatga tcatttattc tgcctcccag agcctgataa aaacggttag cgcttcgtta
     6661 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     6721 taatggtgca gggcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     6781 tgttgggcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     6841 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct accggcagcg gtgcggactg
     6901 ttgtaactca gaataagaaa tgaggccgct catggcgttg actctcagtc atagtatcgt
     6961 ggtatcaccg gttggttcca ctctctgttg cgggcaactt cagcagcacg taggggactt
     7021 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     7081 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     7141 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     7201 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     7261 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     7321 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     7381 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     7441 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     7501 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     7561 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     7621 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     7681 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     7741 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga ag
//
