LOCUS       sequence-347013-        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        1..105
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(106..174,175..966)
                     /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(324..343)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1137..1725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1626..1645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1879..1896
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2013..2034
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2049..2054,2055..2072,2073..2079)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2087..2103
                     /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     2092..2114
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2111..2127
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2111..2127
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7092..7118
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7128..7145
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7163..7292
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     promoter        7375..7774
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        7776..7805
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     primer_bind     complement(7855..7874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
       61 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      121 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      181 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      241 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
      301 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
      361 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
      421 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
      481 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
      541 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
      601 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
      661 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
      721 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
      781 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
      841 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
      901 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
      961 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1021 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1081 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1141 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1201 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1261 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     1321 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1381 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1441 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1501 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1561 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1621 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1681 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1741 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     1801 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     1861 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     1921 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1981 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     2041 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     2101 caatttcaca caggaaacag ctatgaccat gattacgcca agctttgtcg ccggaacgca
     2161 gcgacagaga ttccaatgtg tccgtatctt tcaggctttt gcccttcagt tccagacgaa
     2221 gcgactggcg attcgcgtgt ggggtctgct tcagggtctt gtgaattagg gcgcgcagat
     2281 cgccgatggg cgtggcgccg gagggcacct tcaccttgcc gtacggcttg ctgttcttcg
     2341 cgttcaaaat ctccagctcc attttgcttt cggtgcgctt gcaatcagta ctgtccaaaa
     2401 tcgaaaatcg ccgaaccgta gtgtgaccgt gcggggctct gcgaaaataa acttttttag
     2461 gtatatggcc acacacgggg aaagcacagt ggattatatg ttttaatatt ataatatgca
     2521 ggttttcatt acttatccag atgtaagccc acttaaagcg atttaacaat tatttgccga
     2581 aagagtataa acaaatttca cttaaaaatg gattaagaaa agctagcttg tgtaagatta
     2641 tgcgcagcgt tgccagatag ctccatttaa aacacttcaa aaacaataag ttttgaaaat
     2701 atatacataa atagcagtcg ttgccgcaac gctcaacaca tcacactttt aaaacaccct
     2761 ttacctacac agaattactt tttaaatttc cagtcaagct gcgagtttca aaattatagc
     2821 cggtagagaa gacagtgcta tttcaaaagc aaactaacaa gggtcttaaa ttccaaaaca
     2881 ccaatcctaa caagccttgg acttttgtaa gtttagatca aaggtggcat tgcattcaat
     2941 gtcatggtaa gaagtaggtc gtctaggtag aaatcctcat tcagccggtc aagtcagtac
     3001 gagaaaggtc tcaatttgaa attgtcttaa aaatatttta ttgttttgta ctgtggtgag
     3061 tttaaacgaa aaacacaaaa aaaaagtgat acacagaaat cataaaaaat tttaatacaa
     3121 ggtattcgta cgtatcaaaa acatttcggc acaatttttt ttctctgtac taaagtgtta
     3181 cgaacactac ggtatttttt agtgattttc aacggacacc gaaggtatat aaacagcgtt
     3241 cgcgaacggt cgccttcaaa accaattgac atttgcagca gcaagtacaa gtagaaagta
     3301 aagcgcaatc agcgaaaaat ttatacttaa ttgttggtga ttaaagtaca attaaaagaa
     3361 cattctcgaa agtcacaaga aacgtaagtt tttaactcgc tgttaccaat tagtaataag
     3421 agcaacaaga cgttgagtaa tttcaagaaa aactgcattt caaggtcttt gttcggccat
     3481 ttttttttat tcaacgctct acgtaattac aaaataagaa attggcagcc acgcatcttg
     3541 ttttcccaat gaattggcat caaaacgcaa acaaatctat aaataaaact tgcgtgttga
     3601 ttttcgccaa gatttattgg caaattgtga aattcgcagt gacgcatttg aaaattcgag
     3661 aaatcacgaa cgcactcgat cgagcatttg tgtgcatgtt attagttagt tgtttagtta
     3721 attgaagtat tttaccaacg aaatccactt atttttagct gaaatagagt aggttgctta
     3781 aacaaagcca cgtctgaaaa tttcttattg cttgtagttg tgacgtcacc atatacacac
     3841 aaaataatgt gtatgcatgc atttcagctg tgtatatata catgcacaca ctcgcaacac
     3901 gaaaacgatg acgaagcaac ggaacaaagg tttctcaact accctttgtt ccctgtttct
     3961 tcgctttcct ttgttccaat attcgtagag ggttaatagg ggtttctcaa caaagttggc
     4021 gtcgataaat aagtttccca tttttattcc ccagccagga agttagtttc aatagttttg
     4081 taatttcaac gaaactcatt tgatttcgta ctaattttcc acatctctat tttctgcccg
     4141 cagaataatc caaactgcag gtcgacaaga ccatgattga gaagaagaaa tcctttgcca
     4201 aagggatggg cgtgaagagc actctggtga gcggcagcaa ggtgtacatg accacctttg
     4261 cagaggggag tgatgcccgg ctggagaaga tcgtggaagg ggacagcatc cgctctgtga
     4321 atgaaggaga agctttcagt gcagaaatgg ctgacaagaa tgctgggtac aaaatcggga
     4381 acgccaagtt cagccacccc aagggatatg cagtggttgc taacaatcct ctctacacag
     4441 gacctgtgca gcaggacatg ctggggctga aggagacttt ggagaagcgg tatttcgggg
     4501 agtctgcaga tggaaatgac aatatctgca ttcaggtgat tcataatatt cttgatattg
     4561 aaaagatcct ggctgagtac atcacaaatg ctgcatatgc tgttaataac atctcaggct
     4621 tggacaagga tattattggg tttgggaagt tttccacagt gtatacctac gatgaattta
     4681 aagaccccga gcaccacaga gctgccttca acaataatga caaattgatc aatgccatca
     4741 aggctcagta tgatgaattt gacaacttcc tggacaaccc aaggctgggc tattttggcc
     4801 aggccttctt cagcaaagag ggccgcaact acatcattaa ttacgggaat gaatgttatg
     4861 atatcctcgc cctgctctct ggcctggccc actgggtggt cgccaacaac gaggaagaaa
     4921 gcaggatttc taggacctgg ctgtacaatc ttgacaagaa tctggacaat gaatatatct
     4981 ccaccctcaa ctatttgtat gaccggatca ccaacgaact cacaaacagc ttttctaaga
     5041 actcagccgc aaatgtgaac tacattgcag agacactggg catcaacccc gccgagtttg
     5101 ctgaacagta cttccggttc tccattatga aggagcagaa gaatttgggt tttaacatta
     5161 ctaaacttag ggaggtcatg ctcgacagga aagatatgtc tgagatccgg aagaaccaca
     5221 aggtcttcga ttccatcagg acaaaagtct acaccatgat ggactttgtt atttaccgct
     5281 attacattga agaagatgct aaagtggccg ccgctaataa gagcctgcca gataatgaga
     5341 aatctctcag cgagaaggac atctttgtga tcaacctgcg gggctccttc aatgatgatc
     5401 agaaggatgc actgtactac gatgaagcca accgcatctg gagaaagctg gagaacatca
     5461 tgcacaatat aaaggaattc cgcggcaata agacacggga gtacaagaag aaagacgccc
     5521 cacgcctgcc cagaattctg cctgctggcc gggatgtctc tgccttttca aaactcatgt
     5581 atgccttgac aatgtttctg gatggcaaag aaatcaatga tttgctgaca accctgatca
     5641 acaaatttga caacatccag agcttcctca aggtgatgcc cctgattgga gtaaatgcaa
     5701 aatttgtcga ggagtatgcc ttcttcaagg actctgccaa gattgccgat gagctgcgcc
     5761 tcattaagtc attcgcccgc atgggagagc ccatcgcaga cgcaagaaga gccatgtaca
     5821 tcgatgccat tcgcattttg ggcaccaatc tctcctatga cgagctcaaa gctctggccg
     5881 acaccttctc tttggatgaa aatggcaaca agcttaagaa ggggaaacat ggcatgagaa
     5941 atttcatcat caacaacgtc atttccaaca agcgcttcca ctatctcatc agatatggag
     6001 accctgccca cctgcatgaa attgctaaga atgaggctgt ggtgaaattc gtgctgggaa
     6061 ggatcgccga cattcaaaag aagcaggggc agaatggaaa gaaccagatc gaccgctact
     6121 atgaaacctg tatcggaaaa gataagggca agtctgtcag tgagaaagtg gacgccctca
     6181 ccaagatcat cactggaatg aactatgacc agttcgacaa gaaaagaagt gtgatcgagg
     6241 acacaggccg ggagaatgcc gagagagaga agttcaagaa aattatcagc ctctatctga
     6301 ccgtcatcta ccacatcctg aagaacattg tcaacattaa tgccaggtac gtgattggct
     6361 ttcactgcgt tgaaagagat gcccagcttt ataaggagaa aggctacgac attaacttga
     6421 agaaactgga ggagaaagga tttagcagcg tgaccaaact gtgtgccggc attgacgaga
     6481 ccgcccctga taaacggaaa gatgtggaga aagagatggc cgagcgggcc aaggagagca
     6541 tcgattctct ggaaagtgcc aaccctaagc tctacgccaa ttatattaaa tattctgatg
     6601 agaagaaagc agaagagttc acccggcaga ttaacaggga aaaggccaaa acagccctga
     6661 acgcctacct gagaaacacc aagtggaatg tcatcattag agaagacctg cttcggattg
     6721 ataacaagac ctgcaccctc ttcgccaaca aagccgtggc cttggaagtg gctcgctatg
     6781 tgcatgccta tatcaatgac atcgctgagg tgaactccta cttccagctg tatcactaca
     6841 tcatgcagag aatcattatg aatgaaaggt atgagaaatc atctggtaaa gtatctgaat
     6901 actttgatgc tgtcaatgat gagaagaagt acaacgacag actcctgaag ctgctgtgcg
     6961 tgccctttgg atactgcatc cccaggttta agaacctcag tattgaggcc ctctttgata
     7021 ggaatgaggc agcaaagttt gataaagaaa agaagaaagt ttctggaaac agtggaagtg
     7081 gcgcagctgc ttacccctac gacgtgcctg actacgcctg aaccggtcat catcaccatc
     7141 accattgagt ttatctgact aaatcttagt ttgtattgtc atgttttaat acaatatgtt
     7201 atgtttaaat atgtttttaa taaattttat aaaataattt caacttttat tgtaacaaca
     7261 ttgtccattt acacactcct ttcaagcgcg tgggatcgat gctcactcaa aggcggtaat
     7321 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcca tatggttcga
     7381 cttgcagcct gaaatacggc acgagtagga aaagccgagt caaatgccga atgcagagtc
     7441 tcattacagc acaatcaact caagaaaaac tcgacacttt tttaccattt gcacttaaat
     7501 ccttttttat tcgttatgta tacttttttt ggtccctaac caaaacaaaa ccaaactctc
     7561 ttagtcgtgc ctctatattt aaaactatca atttattata gtcaataaat cgaactgtgt
     7621 tttcaacaaa cgaacaatag gacactttga ttctaaagga aattttgaaa atcttaagca
     7681 gagggttctt aagaccattt gccaattctt ataattctca actgctcttt cctgatgttg
     7741 atcatttata taggtatgtt ttcctcaata cttcgaaccc ctaccaactg gtcggggttt
     7801 gaaacgagac ggttgtattt gtacgtctct ttttttcata tggtgcactc tcagtacaat
     7861 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     7921 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgtcat gataataatg
     7981 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtg
//
