LOCUS       sequence-341533-        8019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses catalytic dead RxCas13d and its associated guide RNA in 
            Drosophila cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8019)
  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 8019)
  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 dRxCas13d + RxCas13d guide RNA
FEATURES             Location/Qualifiers
     source          1..8019
                     /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             7147..7173
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7183..7200
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7218..7347
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     promoter        7430..7829
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        7831..7860
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     primer_bind     complement(7910..7929)
                     /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 cgccaaagct 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 tcccgcagaa
     4201 taatccaaac tgcaggtcga caagaccatg attgagaaga agaaatcctt tgccaaaggg
     4261 atgggcgtga agagcactct ggtgagcggc agcaaggtgt acatgaccac ctttgcagag
     4321 gggagtgatg cccggctgga gaagatcgtg gaaggggaca gcatccgctc tgtgaatgaa
     4381 ggagaagctt tcagtgcaga aatggctgac aagaatgctg ggtacaaaat cgggaacgcc
     4441 aagttcagcc accccaaggg atatgcagtg gttgctaaca atcctctcta cacaggacct
     4501 gtgcagcagg acatgctggg gctgaaggag actttggaga agcggtattt cggggagtct
     4561 gcagatggaa atgacaatat ctgcattcag gtgattcata atattcttga tattgaaaag
     4621 atcctggctg agtacatcac aaatgctgca tatgctgtta ataacatctc aggcttggac
     4681 aaggatatta ttgggtttgg gaagttttcc acagtgtata cctacgatga atttaaagac
     4741 cccgagcacc acagagctgc cttcaacaat aatgacaaat tgatcaatgc catcaaggct
     4801 cagtatgatg aatttgacaa cttcctggac aacccaaggc tgggctattt tggccaggcc
     4861 ttcttcagca aagagggccg caactacatc attaattacg ggaatgaatg ttatgatatc
     4921 ctcgccctgc tctctggcct ggcccactgg gtggtcgcca acaacgagga agaaagcagg
     4981 atttctagga cctggctgta caatcttgac aagaatctgg acaatgaata tatctccacc
     5041 ctcaactatt tgtatgaccg gatcaccaac gaactcacaa acagcttttc taagaactca
     5101 gccgcaaatg tgaactacat tgcagagaca ctgggcatca accccgccga gtttgctgaa
     5161 cagtacttcc ggttctccat tatgaaggag cagaagaatt tgggttttaa cattactaaa
     5221 cttagggagg tcatgctcga caggaaagat atgtctgaga tccggaagaa ccacaaggtc
     5281 ttcgattcca tcaggacaaa agtctacacc atgatggact ttgttattta ccgctattac
     5341 attgaagaag atgctaaagt ggccgccgct aataagagcc tgccagataa tgagaaatct
     5401 ctcagcgaga aggacatctt tgtgatcaac ctgcggggct ccttcaatga tgatcagaag
     5461 gatgcactgt actacgatga agccaaccgc atctggagaa agctggagaa catcatgcac
     5521 aatataaagg aattccgcgg caataagaca cgggagtaca agaagaaaga cgccccacgc
     5581 ctgcccagaa ttctgcctgc tggccgggat gtctctgcct tttcaaaact catgtatgcc
     5641 ttgacaatgt ttctggatgg caaagaaatc aatgatttgc tgacaaccct gatcaacaaa
     5701 tttgacaaca tccagagctt cctcaaggtg atgcccctga ttggagtaaa tgcaaaattt
     5761 gtcgaggagt atgccttctt caaggactct gccaagattg ccgatgagct gcgcctcatt
     5821 aagtcattcg cccgcatggg agagcccatc gcagacgcaa gaagagccat gtacatcgat
     5881 gccattcgca ttttgggcac caatctctcc tatgacgagc tcaaagctct ggccgacacc
     5941 ttctctttgg atgaaaatgg caacaagctt aagaagggga aacatggcat gagaaatttc
     6001 atcatcaaca acgtcatttc caacaagcgc ttccactatc tcatcagata tggagaccct
     6061 gcccacctgc atgaaattgc taagaatgag gctgtggtga aattcgtgct gggaaggatc
     6121 gccgacattc aaaagaagca ggggcagaat ggaaagaacc agatcgaccg ctactatgaa
     6181 acctgtatcg gaaaagataa gggcaagtct gtcagtgaga aagtggacgc cctcaccaag
     6241 atcatcactg gaatgaacta tgaccagttc gacaagaaaa gaagtgtgat cgaggacaca
     6301 ggccgggaga atgccgagag agagaagttc aagaaaatta tcagcctcta tctgaccgtc
     6361 atctaccaca tcctgaagaa cattgtcaac attaatgcca ggtacgtgat tggctttcac
     6421 tgcgttgaaa gagatgccca gctttataag gagaaaggct acgacattaa cttgaagaaa
     6481 ctggaggaga aaggatttag cagcgtgacc aaactgtgtg ccggcattga cgagaccgcc
     6541 cctgataaac ggaaagatgt ggagaaagag atggccgagc gggccaagga gagcatcgat
     6601 tctctggaaa gtgccaaccc taagctctac gccaattata ttaaatattc tgatgagaag
     6661 aaagcagaag agttcacccg gcagattaac agggaaaagg ccaaaacagc cctgaacgcc
     6721 tacctgagaa acaccaagtg gaatgtcatc attagagaag acctgcttcg gattgataac
     6781 aagacctgca ccctcttcgc caacaaagcc gtggccttgg aagtggctcg ctatgtgcat
     6841 gcctatatca atgacatcgc tgaggtgaac tcctacttcc agctgtatca ctacatcatg
     6901 cagagaatca ttatgaatga aaggtatgag aaatcatctg gtaaagtatc tgaatacttt
     6961 gatgctgtca atgatgagaa gaagtacaac gacagactcc tgaagctgct gtgcgtgccc
     7021 tttggatact gcatccccag gtttaagaac ctcagtattg aggccctctt tgataggaat
     7081 gaggcagcaa agtttgataa agaaaagaag aaagtttctg gaaacagtgg aagtggcgca
     7141 gctgcttacc cctacgacgt gcctgactac gcctgaaccg gtcatcatca ccatcaccat
     7201 tgagtttatc tgactaaatc ttagtttgta ttgtcatgtt ttaatacaat atgttatgtt
     7261 taaatatgtt tttaataaat tttataaaat aatttcaact tttattgtaa caacattgtc
     7321 catttacaca ctcctttcaa gcgcgtggga tcgatgctca ctcaaaggcg gtaatacggt
     7381 tatccacaga atcaggggat aacgcaggaa agaacatgtg agccatatgg ttcgacttgc
     7441 agcctgaaat acggcacgag taggaaaagc cgagtcaaat gccgaatgca gagtctcatt
     7501 acagcacaat caactcaaga aaaactcgac acttttttac catttgcact taaatccttt
     7561 tttattcgtt atgtatactt tttttggtcc ctaaccaaaa caaaaccaaa ctctcttagt
     7621 cgtgcctcta tatttaaaac tatcaattta ttatagtcaa taaatcgaac tgtgttttca
     7681 acaaacgaac aataggacac tttgattcta aaggaaattt tgaaaatctt aagcagaggg
     7741 ttcttaagac catttgccaa ttcttataat tctcaactgc tctttcctga tgttgatcat
     7801 ttatataggt atgttttcct caatacttcg aacccctacc aactggtcgg ggtttgaaac
     7861 gagacggttg tatttgtacg tctctttttt tcatatggtg cactctcagt acaatctgct
     7921 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     7981 gggcttgtct gctcccggca tccgcttaca gacaagctg
//
