LOCUS       sequence-346369-        8665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LwaCas13a and its associated guide RNA in Drosophila 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8665)
  AUTHORS   Ai Y, Liang D, Wilusz JE
  TITLE     CRISPR/Cas13 effectors have differing extents of off-target effects 
            that limit their utility in eukaryotic cells.
  JOURNAL   Nucleic Acids Res. 2022 Mar 4. pii: 6542487. doi: 
            10.1093/nar/gkac159.
  PUBMED    35244715
REFERENCE   2  (bases 1 to 8665)
  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: "Nucleic 
            Acids Res. 2022 Mar 4. pii: 6542487. doi: 10.1093/nar/gkac159."
COMMENT     Sequence Label: pUb LwaCas13a + LwaCas13a Guide RNA
FEATURES             Location/Qualifiers
     source          1..8665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        56..160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(161..229,230..1021)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(379..398)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1192..1780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1681..1700
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1934..1951
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2068..2089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2104..2109,2110..2127,2128..2134)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2142..2158
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2147..2169
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2166..2182
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2166..2182
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4243..4308
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4321..7773
                     /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="KVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYI
                     KNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYD
                     LKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENN
                     VEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKY
                     KIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDK
                     EELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLN
                     KLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETEN
                     ENGITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEI
                     EDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKI
                     FKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWS
                     VPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQK
                     FENIEKTVPVEYLAIIQSRGMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIES
                     NNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYT
                     ENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFE
                     LEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKI
                     ADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIG
                     NIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEI
                     FNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYI
                     AHFNYIPHAEVSLLEVLENLRKLLSYGRKLKNAIMKSIVDILKEYGFVATFKIGADKKI
                     EIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
     CDS             7783..7809
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7819..7836
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7854..7983
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     promoter        8066..8465
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     primer_bind     complement(8556..8575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
       61 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
      121 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
      181 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
      241 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
      301 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
      361 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
      421 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
      481 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
      541 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
      601 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
      661 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
      721 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      781 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      841 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
      901 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      961 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     1021 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     1081 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     1141 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1201 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1261 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1321 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1381 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1441 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1501 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1561 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1621 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1681 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1741 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1801 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1861 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1921 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     1981 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2041 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     2101 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     2161 tcacacagga aacagctatg accatgatta cgccaagctt tgtcgccgga acgcagcgac
     2221 agagattcca atgtgtccgt atctttcagg cttttgccct tcagttccag acgaagcgac
     2281 tggcgattcg cgtgtggggt ctgcttcagg gtcttgtgaa ttagggcgcg cagatcgccg
     2341 atgggcgtgg cgccggaggg caccttcacc ttgccgtacg gcttgctgtt cttcgcgttc
     2401 aaaatctcca gctccatttt gctttcggtg cgcttgcaat cagtactgtc caaaatcgaa
     2461 aatcgccgaa ccgtagtgtg accgtgcggg gctctgcgaa aataaacttt tttaggtata
     2521 tggccacaca cggggaaagc acagtggatt atatgtttta atattataat atgcaggttt
     2581 tcattactta tccagatgta agcccactta aagcgattta acaattattt gccgaaagag
     2641 tataaacaaa tttcacttaa aaatggatta agaaaagcta gcttgtgtaa gattatgcgc
     2701 agcgttgcca gatagctcca tttaaaacac ttcaaaaaca ataagttttg aaaatatata
     2761 cataaatagc agtcgttgcc gcaacgctca acacatcaca cttttaaaac accctttacc
     2821 tacacagaat tactttttaa atttccagtc aagctgcgag tttcaaaatt atagccggta
     2881 gagaagacag tgctatttca aaagcaaact aacaagggtc ttaaattcca aaacaccaat
     2941 cctaacaagc cttggacttt tgtaagttta gatcaaaggt ggcattgcat tcaatgtcat
     3001 ggtaagaagt aggtcgtcta ggtagaaatc ctcattcagc cggtcaagtc agtacgagaa
     3061 aggtctcaat ttgaaattgt cttaaaaata ttttattgtt ttgtactgtg gtgagtttaa
     3121 acgaaaaaca caaaaaaaaa gtgatacaca gaaatcataa aaaattttaa tacaaggtat
     3181 tcgtacgtat caaaaacatt tcggcacaat tttttttctc tgtactaaag tgttacgaac
     3241 actacggtat tttttagtga ttttcaacgg acaccgaagg tatataaaca gcgttcgcga
     3301 acggtcgcct tcaaaaccaa ttgacatttg cagcagcaag tacaagtaga aagtaaagcg
     3361 caatcagcga aaaatttata cttaattgtt ggtgattaaa gtacaattaa aagaacattc
     3421 tcgaaagtca caagaaacgt aagtttttaa ctcgctgtta ccaattagta ataagagcaa
     3481 caagacgttg agtaatttca agaaaaactg catttcaagg tctttgttcg gccatttttt
     3541 tttattcaac gctctacgta attacaaaat aagaaattgg cagccacgca tcttgttttc
     3601 ccaatgaatt ggcatcaaaa cgcaaacaaa tctataaata aaacttgcgt gttgattttc
     3661 gccaagattt attggcaaat tgtgaaattc gcagtgacgc atttgaaaat tcgagaaatc
     3721 acgaacgcac tcgatcgagc atttgtgtgc atgttattag ttagttgttt agttaattga
     3781 agtattttac caacgaaatc cacttatttt tagctgaaat agagtaggtt gcttaaacaa
     3841 agccacgtct gaaaatttct tattgcttgt agttgtgacg tcaccatata cacacaaaat
     3901 aatgtgtatg catgcatttc agctgtgtat atatacatgc acacactcgc aacacgaaaa
     3961 cgatgacgaa gcaacggaac aaaggtttct caactaccct ttgttccctg tttcttcgct
     4021 ttcctttgtt ccaatattcg tagagggtta ataggggttt ctcaacaaag ttggcgtcga
     4081 taaataagtt tcccattttt attccccagc caggaagtta gtttcaatag ttttgtaatt
     4141 tcaacgaaac tcatttgatt tcgtactaat tttccacatc tctattttct gcccgcagaa
     4201 taatccaaac tgcaggtcga ctctagctag aggaagctta tggactacaa agaccatgac
     4261 ggtgattata aagatcatga tatcgattac aaggatgacg atgacaagga tccactagtc
     4321 aaagtgacca aggtcgacgg catcagccac aagaagtaca tcgaagaggg caagctcgtg
     4381 aagtccacca gcgaggaaaa ccggaccagc gagagactga gcgagctgct gagcatccgg
     4441 ctggacatct acatcaagaa ccccgacaac gcctccgagg aagagaaccg gatcagaaga
     4501 gagaacctga agaagttctt tagcaacaag gtgctgcacc tgaaggacag cgtgctgtat
     4561 ctgaagaacc ggaaagaaaa gaacgccgtg caggacaaga actatagcga agaggacatc
     4621 agcgagtacg acctgaaaaa caagaacagc ttctccgtgc tgaagaagat cctgctgaac
     4681 gaggacgtga actctgagga actggaaatc tttcggaagg acgtggaagc caagctgaac
     4741 aagatcaaca gcctgaagta cagcttcgaa gagaacaagg ccaactacca gaagatcaac
     4801 gagaacaacg tggaaaaagt gggcggcaag agcaagcgga acatcatcta cgactactac
     4861 agagagagcg ccaagcgcaa cgactacatc aacaacgtgc aggaagcctt cgacaagctg
     4921 tataagaaag aggatatcga gaaactgttt ttcctgatcg agaacagcaa gaagcacgag
     4981 aagtacaaga tccgcgagta ctatcacaag atcatcggcc ggaagaacga caaagagaac
     5041 ttcgccaaga ttatctacga agagatccag aacgtgaaca acatcaaaga gctgattgag
     5101 aagatccccg acatgtctga gctgaagaaa agccaggtgt tctacaagta ctacctggac
     5161 aaagaggaac tgaacgacaa gaatattaag tacgccttct gccacttcgt ggaaatcgag
     5221 atgtcccagc tgctgaaaaa ctacgtgtac aagcggctga gcaacatcag caacgataag
     5281 atcaagcgga tcttcgagta ccagaatctg aaaaagctga tcgaaaacaa actgctgaac
     5341 aagctggaca cctacgtgcg gaactgcggc aagtacaact actatctgca agtgggcgag
     5401 atcgccacct ccgactttat cgcccggaac cggcagaacg aggccttcct gagaaacatc
     5461 atcggcgtgt ccagcgtggc ctacttcagc ctgaggaaca tcctggaaac cgagaacgag
     5521 aacggtatca ccggccggat gcggggcaag accgtgaaga acaacaaggg cgaagagaaa
     5581 tacgtgtccg gcgaggtgga caagatctac aatgagaaca agcagaacga agtgaaagaa
     5641 aatctgaaga tgttctacag ctacgacttc aacatggaca acaagaacga gatcgaggac
     5701 ttcttcgcca acatcgacga ggccatcagc agcatcagac acggcatcgt gcacttcaac
     5761 ctggaactgg aaggcaagga catcttcgcc ttcaagaata tcgcccccag cgagatctcc
     5821 aagaagatgt ttcagaacga aatcaacgaa aagaagctga agctgaaaat cttcaagcag
     5881 ctgaacagcg ccaacgtgtt caactactac gagaaggatg tgatcatcaa gtacctgaag
     5941 aataccaagt tcaacttcgt gaacaaaaac atccccttcg tgcccagctt caccaagctg
     6001 tacaacaaga ttgaggacct gcggaatacc ctgaagtttt tttggagcgt gcccaaggac
     6061 aaagaagaga aggacgccca gatctacctg ctgaagaata tctactacgg cgagttcctg
     6121 aacaagttcg tgaaaaactc caaggtgttc tttaagatca ccaatgaagt gatcaagatt
     6181 aacaagcagc ggaaccagaa aaccggccac tacaagtatc agaagttcga gaacatcgag
     6241 aaaaccgtgc ccgtggaata cctggccatc atccagagca gagggatgat caacaaccag
     6301 gacaaagagg aaaagaatac ctacatcgac tttattcagc agattttcct gaagggcttc
     6361 atcgactacc tgaacaagaa caatctgaag tatatcgaga gcaacaacaa caatgacaac
     6421 aacgacatct tctccaagat caagatcaaa aaggataaca aagagaagta cgacaagatc
     6481 ctgaagaact atgagaagca caatcggaac aaagaaatcc ctcacgagat caatgagttc
     6541 gtgcgcgaga tcaagctggg gaagattctg aagtacaccg agaatctgaa catgttttac
     6601 ctgatcctga agctgctgaa ccacaaagag ctgaccaacc tgaagggcag cctggaaaag
     6661 taccagtccg ccaacaaaga agaaaccttc agcgacgagt tggaactgat caacctgctg
     6721 aacctggaca acaacagagt gaccgaggac ttcgagctgg aagccaacga gatcggcaag
     6781 ttcctggact tcaacgaaaa caaaatcaag gaccggaaag agctgaaaaa gttcgacacc
     6841 aacaagatct atttcgacgg cgagaacatc atcaagcacc gggccttcta caatatcaag
     6901 aaatacggca tgctgaatct gctggaaaag atcgccgata aggccaagta taagatcagc
     6961 ctgaaagaac tgaaagagta cagcaacaag aagaatgaga ttgaaaagaa ctacaccatg
     7021 cagcagaacc tgcaccggaa gtacgccaga cccaagaagg acgaaaagtt caacgacgag
     7081 gactacaaag agtatgagaa ggccatcggc aacatccaga agtacaccca cctgaagaac
     7141 aaggtggaat tcaatgagct gaacctgctg cagggcctgc tgctgaagat cctgcaccgg
     7201 ctcgtgggct acaccagcat ctgggagcgg gacctgagat tccggctgaa gggcgagttt
     7261 cccgagaacc actacatcga ggaaattttc aatttcgaca actccaagaa tgtgaagtac
     7321 aaaagcggcc agatcgtgga aaagtatatc aacttctaca aagaactgta caaggacaat
     7381 gtggaaaagc ggagcatcta ctccgacaag aaagtgaaga aactgaagca ggaaaaaaag
     7441 gacctgtaca tccggaacta cattgcccac ttcaactaca tcccccacgc cgaggttagc
     7501 ctgctggaag tgctggaaaa cctgcggaag ctgctgtcct acggccggaa gctgaagaac
     7561 gccatcatga agtccatcgt ggacattctg aaagaatacg gcttcgtggc caccttcaag
     7621 atcggcgctg acaagaagat cgaaatccag accctggaat cagagaagat cgtgcacctg
     7681 aagaatctga agaaaaagaa actgatgacc gaccggaaca gcgaggaact gtgcgaactc
     7741 gtgaaagtca tgttcgagta caaggccctg gaaggaagtg gctaccccta cgacgtgcct
     7801 gactacgcct gaaccggtca tcatcaccat caccattgag tttatctgac taaatcttag
     7861 tttgtattgt catgttttaa tacaatatgt tatgtttaaa tatgttttta ataaatttta
     7921 taaaataatt tcaactttta ttgtaacaac attgtccatt tacacactcc tttcaagcgc
     7981 gtgggatcga tgctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     8041 caggaaagaa catgtgagcc atatggttcg acttgcagcc tgaaatacgg cacgagtagg
     8101 aaaagccgag tcaaatgccg aatgcagagt ctcattacag cacaatcaac tcaagaaaaa
     8161 ctcgacactt ttttaccatt tgcacttaaa tcctttttta ttcgttatgt atactttttt
     8221 tggtccctaa ccaaaacaaa accaaactct cttagtcgtg cctctatatt taaaactatc
     8281 aatttattat agtcaataaa tcgaactgtg ttttcaacaa acgaacaata ggacactttg
     8341 attctaaagg agattttgaa aatcttaagc agagggttct taagaccatt tgccaattct
     8401 tataattctc aactgctctt tcctgatgtt gatcatttat ataggtatgt tttcctcaat
     8461 acttcggatt tagactaccc caaaaacgaa ggggactaaa acgagacggt tgtatttgta
     8521 gttgcgtctc tttttttcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     8581 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     8641 ccggcatccg cttacagaca agctg
//
