LOCUS       sequence-218064-        7187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  OsActin Promoter-LwaCas13a-HSP Terminator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7187)
  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 7187)
  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: pJJB302
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        801..1642
                     /label=Act1 promoter
                     /note="rice actin promoter"
     CDS             1645..1662
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2230..5685
                     /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
                     NENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE
                     IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
                     IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
                     SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
                     KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
                     SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
                     TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
                     ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
                     IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
                     GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
                     IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY
                     IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
                     IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
     terminator      5696..5944
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        6016..6120
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct actaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgctcacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccacc
      781 tgctcatgat cggcgcgccg tcgaggtcat tcatatgctt gagaagagag tcgggatagt
      841 ccaaaataaa acaaaggtaa gattacctgg tcaaaagtga aaacatcagt taaaaggtgg
      901 tataaagtaa aatatcggta ataaaaggtg gcccaaagtg aaatttactc ttttctacta
      961 ttataaaaat tgaggatgtt tttgtcggta ctttgatacg tcatttttgt atgaattggt
     1021 ttttaagttt attcgctttt ggaaatgcat atctgtattt gagtcgggtt ttaagttcgt
     1081 ttgcttttgt aaatacagag ggatttgtat aagaaatatc tttaaaaaaa cccatatgct
     1141 aatttgacat aatttttgag aaaaatatat attcaggcga attctcacaa tgaacaataa
     1201 taagattaaa atagctttcc cccgttgcag cgcatgggta ttttttctag taaaaataaa
     1261 agataaactt agactcaaaa catttacaaa aacaacccct aaagttccta aagcccaaag
     1321 tgctatccac gatccatagc aagcccagcc caacccaacc caacccaacc caccccagtc
     1381 cagccaactg gacaatagtc tccacacccc cccactatca ccgtgagttg tccgcacgca
     1441 ccgcacgtct cgcagccaaa aaaaaaaaaa gaaagaaaaa aaagaaaaag aaaaaacagc
     1501 agggggtccg ggtcgtgggg gccggaaacg cgaggaggat cgcgagccag cgacgaggcc
     1561 ggccctccct ccgcttccaa agaaacgccc cccatcgcca ctatatacat acccccccct
     1621 ctcctcccat ccccccaacc ctaccaccac caccaccacc acctccacct cctcccccct
     1681 cgctgccgga cgacgagctc ctcccccctc cccctccgcc gccgccgcgc cggtaaccac
     1741 cccgcccctc tcctctttct ttctccgttt tttttttccg tctcggtctc gatctttggc
     1801 cttggtagtt tgggtgggcg agaggcggct tcgtgcgcgc ccagatcggt gcgcgggagg
     1861 ggcgggatct cgcggctggg gctctcgccg gcgtggatcc ggcccggatc tcgcggggaa
     1921 tggggctctc ggatgtagat ctgcgatccg ccgttgttgg gggagatgat ggggggttta
     1981 aaatttccgc catgctaaac aagatcagga agaggggaaa agggcactat ggtttatatt
     2041 tttatatatt tctgctgctt cgtcaggctt agatgtgcta gatctttctt tcttcttttt
     2101 gtgggtagaa tttgaatccc tcagcattgt tcatcggtag tttttctttt catgatttgt
     2161 gacaaatgca gcctcgtgcg gagctttttt gtaggtagac gataagcttc ctgcatcgac
     2221 atgggatcca tgaaagtgac caaggtcgac ggcatcagcc acaagaagta catcgaagag
     2281 ggcaagctcg tgaagtccac cagcgaggaa aaccggacca gcgagagact gagcgagctg
     2341 ctgagcatcc ggctggacat ctacatcaag aaccccgaca acgcctccga ggaagagaac
     2401 cggatcagaa gagagaacct gaagaagttc tttagcaaca aggtgctgca cctgaaggac
     2461 agcgtgctgt atctgaagaa ccggaaagaa aagaacgccg tgcaggacaa gaactatagc
     2521 gaagaggaca tcagcgagta cgacctgaaa aacaagaaca gcttctccgt gctgaagaag
     2581 atcctgctga acgaggacgt gaactctgag gaactggaaa tctttcggaa ggacgtggaa
     2641 gccaagctga acaagatcaa cagcctgaag tacagcttcg aagagaacaa ggccaactac
     2701 cagaagatca acgagaacaa cgtggaaaaa gtgggcggca agagcaagcg gaacatcatc
     2761 tacgactact acagagagag cgccaagcgc aacgactaca tcaacaacgt gcaggaagcc
     2821 ttcgacaagc tgtataagaa agaggatatc gagaaactgt ttttcctgat cgagaacagc
     2881 aagaagcacg agaagtacaa gatccgcgag tactatcaca agatcatcgg ccggaagaac
     2941 gacaaagaga acttcgccaa gattatctac gaagagatcc agaacgtgaa caacatcaaa
     3001 gagctgattg agaagatccc cgacatgtct gagctgaaga aaagccaggt gttctacaag
     3061 tactacctgg acaaagagga actgaacgac aagaatatta agtacgcctt ctgccacttc
     3121 gtggaaatcg agatgtccca gctgctgaaa aactacgtgt acaagcggct gagcaacatc
     3181 agcaacgata agatcaagcg gatcttcgag taccagaatc tgaaaaagct gatcgaaaac
     3241 aaactgctga acaagctgga cacctacgtg cggaactgcg gcaagtacaa ctactatctg
     3301 caagtgggcg agatcgccac ctccgacttt atcgcccgga accggcagaa cgaggccttc
     3361 ctgagaaaca tcatcggcgt gtccagcgtg gcctacttca gcctgaggaa catcctggaa
     3421 accgagaacg agaacggtat caccggccgg atgcggggca agaccgtgaa gaacaacaag
     3481 ggcgaagaga aatacgtgtc cggcgaggtg gacaagatct acaatgagaa caagcagaac
     3541 gaagtgaaag aaaatctgaa gatgttctac agctacgact tcaacatgga caacaagaac
     3601 gagatcgagg acttcttcgc caacatcgac gaggccatca gcagcatcag acacggcatc
     3661 gtgcacttca acctggaact ggaaggcaag gacatcttcg ccttcaagaa tatcgccccc
     3721 agcgagatct ccaagaagat gtttcagaac gaaatcaacg aaaagaagct gaagctgaaa
     3781 atcttcaagc agctgaacag cgccaacgtg ttcaactact acgagaagga tgtgatcatc
     3841 aagtacctga agaataccaa gttcaacttc gtgaacaaaa acatcccctt cgtgcccagc
     3901 ttcaccaagc tgtacaacaa gattgaggac ctgcggaata ccctgaagtt tttttggagc
     3961 gtgcccaagg acaaagaaga gaaggacgcc cagatctacc tgctgaagaa tatctactac
     4021 ggcgagttcc tgaacaagtt cgtgaaaaac tccaaggtgt tctttaagat caccaatgaa
     4081 gtgatcaaga ttaacaagca gcggaaccag aaaaccggcc actacaagta tcagaagttc
     4141 gagaacatcg agaaaaccgt gcccgtggaa tacctggcca tcatccagag cagagagatg
     4201 atcaacaacc aggacaaaga ggaaaagaat acctacatcg actttattca gcagattttc
     4261 ctgaagggct tcatcgacta cctgaacaag aacaatctga agtatatcga gagcaacaac
     4321 aacaatgaca acaacgacat cttctccaag atcaagatca aaaaggataa caaagagaag
     4381 tacgacaaga tcctgaagaa ctatgagaag cacaatcgga acaaagaaat ccctcacgag
     4441 atcaatgagt tcgtgcgcga gatcaagctg gggaagattc tgaagtacac cgagaatctg
     4501 aacatgtttt acctgatcct gaagctgctg aaccacaaag agctgaccaa cctgaagggc
     4561 agcctggaaa agtaccagtc cgccaacaaa gaagaaacct tcagcgacga gttggaactg
     4621 atcaacctgc tgaacctgga caacaacaga gtgaccgagg acttcgagct ggaagccaac
     4681 gagatcggca agttcctgga cttcaacgaa aacaaaatca aggaccggaa agagctgaaa
     4741 aagttcgaca ccaacaagat ctatttcgac ggcgagaaca tcatcaagca ccgggccttc
     4801 tacaatatca agaaatacgg catgctgaat ctgctggaaa agatcgccga taaggccaag
     4861 tataagatca gcctgaaaga actgaaagag tacagcaaca agaagaatga gattgaaaag
     4921 aactacacca tgcagcagaa cctgcaccgg aagtacgcca gacccaagaa ggacgaaaag
     4981 ttcaacgacg aggactacaa agagtatgag aaggccatcg gcaacatcca gaagtacacc
     5041 cacctgaaga acaaggtgga attcaatgag ctgaacctgc tgcagggcct gctgctgaag
     5101 atcctgcacc ggctcgtggg ctacaccagc atctgggagc gggacctgag attccggctg
     5161 aagggcgagt ttcccgagaa ccactacatc gaggaaattt tcaatttcga caactccaag
     5221 aatgtgaagt acaaaagcgg ccagatcgtg gaaaagtata tcaacttcta caaagaactg
     5281 tacaaggaca atgtggaaaa gcggagcatc tactccgaca agaaagtgaa gaaactgaag
     5341 caggaaaaaa aggacctgta catccggaac tacattgccc acttcaacta catcccccac
     5401 gccgagatta gcctgctgga agtgctggaa aacctgcgga agctgctgtc ctacgaccgg
     5461 aagctgaaga acgccatcat gaagtccatc gtggacattc tgaaagaata cggcttcgtg
     5521 gccaccttca agatcggcgc tgacaagaag atcgaaatcc agaccctgga atcagagaag
     5581 atcgtgcacc tgaagaatct gaagaaaaag aaactgatga ccgaccggaa cagcgaggaa
     5641 ctgtgcgaac tcgtgaaagt catgttcgag tacaaggccc tggaatgact cgagatatga
     5701 agatgaagat gaaatatttg gtgtgtcaaa taaaaagctt gtgtgcttaa gtttgtgttt
     5761 ttttcttggc ttgttgtgtt atgaatttgt ggctttttct aatattaaat gaatgtaaga
     5821 tcacattata atgaataaac aaatgtttct ataatccatt gtgaatgttt tgttggatct
     5881 cttctgcagc atataactac tgtatgtgct atggtatgga ctatggaata tgattaaaga
     5941 taaggagctc cggtgacgga cgtgggcagg tgatgaaaag ccttagggtc aggtggcact
     6001 tttcggggaa atgtgcgatg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6061 tatccgctaa tgagacaata accctgataa atgcttcaat tatattgaaa aaggaagagt
     6121 atgagtatcc aacatttccg tgtcgccctt attccctttt ttgcggcctt ctgccttcct
     6181 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcc
     6241 cgagtgggtt acattgaact ggatctcaac agcggcaaaa tcttagagag ttttcgcccc
     6301 gaagaacgct tcccaatgat gagcactttc aaagttctgc tatgtggcgc ggtgttatcc
     6361 cgtattgatg ctgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6421 gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6481 tgtagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatt
     6541 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6601 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6661 cctgtagcaa tggcaacaac tctccgcaaa ctattaactg gcgaactact tactcttgct
     6721 tcacgccagc aactcattga ctggatggag gcggacaaag ttgcaggacc acttctgcgc
     6781 tcggcacttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6841 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6901 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6961 tcactgatta agcattggta actgtcagac gaagggaaat aaatagtagc ccgcctgatg
     7021 tgcgggcttt tttttggtgg tggttaccag tgtcgcttag ctaccgtagg tcctcttagg
     7081 cggtccgatt agcgacatag tcatcaatgc cttgacggca aaatccctta acgtgagttt
     7141 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
