LOCUS       sequence-347012-        8019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 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 RxCas13d + RxCas13d guide RNA
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..47)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(201..789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(281..300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(960..1751,1752..1820))
                     /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     1583..1602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1821..1925)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2071..2090
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        complement(2140..2169)
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        complement(2171..2570)
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     polyA_signal    2653..2782
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     CDS             complement(2800..2817)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2827..2853)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2872..5772)
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     primer_bind     complement(7818..7834)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7818..7834)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7831..7853)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7842..7858
                     /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)."
     promoter        complement(join(7866..7872,7873..7890,7891..7896))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7911..7932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
       61 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      361 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      601 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
      661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
      781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
      841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
      901 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
      961 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1021 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1081 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     1141 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1201 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1261 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     1321 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     1381 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     1441 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     1501 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     1561 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     1621 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     1681 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     1741 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     1801 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     1861 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     1921 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     1981 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2041 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     2101 accatatgaa aaaaagagac gtacaaatac aaccgtctcg tttcaaaccc cgaccagttg
     2161 gtaggggttc gaagtattga ggaaaacata cctatataaa tgatcaacat caggaaagag
     2221 cagttgagaa ttataagaat tggcaaatgg tcttaagaac cctctgctta agattttcaa
     2281 aatttccttt agaatcaaag tgtcctattg ttcgtttgtt gaaaacacag ttcgatttat
     2341 tgactataat aaattgatag ttttaaatat agaggcacga ctaagagagt ttggttttgt
     2401 tttggttagg gaccaaaaaa agtatacata acgaataaaa aaggatttaa gtgcaaatgg
     2461 taaaaaagtg tcgagttttt cttgagttga ttgtgctgta atgagactct gcattcggca
     2521 tttgactcgg cttttcctac tcgtgccgta tttcaggctg caagtcgaac catatggctc
     2581 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2641 gagcatcgat cccacgcgct tgaaaggagt gtgtaaatgg acaatgttgt tacaataaaa
     2701 gttgaaatta ttttataaaa tttattaaaa acatatttaa acataacata ttgtattaaa
     2761 acatgacaat acaaactaag atttagtcag ataaactcaa tggtgatggt gatgatgacc
     2821 ggttcaggcg tagtcaggca cgtcgtaggg gtaagcagct gcgccacttc cactgtttcc
     2881 agaaactttc ttcttttctt tatcaaactt tgctgcctca ttcctatcaa agagggcctc
     2941 aatactgagg ttcttaaacc tggggatgca gtatccaaag ggcacgcaca gcagcttcag
     3001 gagtctgtcg ttgtacttct tctcatcatt gacagcatca aagtattcag atactttacc
     3061 agatgatttc tcataccttt cattcataat gattctctgc atgatgtagt gatacagctg
     3121 gaagtaggag ttcacctcag cgatgtcatt gatataggca tgcacatagc gagccacttc
     3181 caagtgcacg gctttgttcc tgaagagggt gcaggtcttg ttatcaatcc gaagcaggtc
     3241 ttctctaatg atgacattcc acttggtgtt tctcaggtag gcgttcaggg ctgttttggc
     3301 cttttccctg ttaatctgcc gggtgaactc ttctgctttc ttctcatcag aatatttaat
     3361 ataattggcg tagagcttag ggttggcact ttccagagaa tcgatgctct ccttggcccg
     3421 ctcggccatc tctttctcca catctttccg tttatcaggg gcggtctcgt caatgccggc
     3481 acacagtttg gtcacgctgc taaatccttt ctcctccagt ttcttcaagt taatgtcgta
     3541 gcctttctcc ttataaagct gggcatctct ttcaacgcag tgaaagccaa tcacgtacct
     3601 ggcattaatg ttgacaatgt tcttcaggat gtggtagatg acggtcagat agaggctgat
     3661 aattttcttg aacttctctc tctcggcatt ctcccggcct gtgtcctcga tcacacttct
     3721 tttcttgtcg aactggtcat agttcattcc agtgatgatc ttggtgaggg cgtccacttt
     3781 ctcactgaca gacttgccct tatcttttcc gatacaggtt tcatagtagc ggtcgatctg
     3841 gttctttcca ttctgcccct gcttcttttg aatgtcggcg atccttccca gcacaaattt
     3901 caccacagcc tcattcttag caatttcatg caggtgggca gggtctccat atctgatgag
     3961 atagtggaag cgcttgttgg aaatgacgtt gttgatgatg aaatttctca tgccatgttt
     4021 ccccttctta agcttgttgc cattttcatc caaagagaag gtgtcggcca gagctttgag
     4081 ctcgtcatag gagagattgg tgcccaaaat gcgaatggca tcgatgtaca tggctcttct
     4141 tgcgtctgcg atgggctctc ccatgcgggc gaatgactta atgaggcgca gctcatcggc
     4201 aatcttggca gagtccttga agaaggcata ctcctcgaca aattttgcat ttactccaat
     4261 caggggcatc accttgagga agctctggat gttgtcaaat ttgttgatca gggttgtcag
     4321 caaatcattg atttctttgc catccagaaa cattgtcaag gcatacatga gttttgaaaa
     4381 ggcagagaca tcccggccag caggcagaat tctgggcagg cgtggggcgt ctttcttctt
     4441 gtactcccgt gtcttattgc cgcggaattc ctttatattg tgcatgatgt tctccagctt
     4501 tctccagatg cggttggctt catcgtagta cagtgcatcc ttctgatcat cattgaagga
     4561 gccccgcagg ttgatcacaa agatgtcctt ctcgctgaga gatttctcat tatctggcag
     4621 gctcttatta gcggcggcca ctttagcatc ttcttcaatg taatagcggt aaataacaaa
     4681 gtccatcatg gtgtagactt ttgtcctgat ggaatcgaag accttgtggt tcttccggat
     4741 ctcagacata tctttcctgt cgagcatgac ctccctaagt ttagtaatgt taaaacccaa
     4801 attcttctgc tccttcataa tggagaaccg gaagtactgt tcagcaaact cggcggggtt
     4861 gatgcccagt gtctctgcaa tgtagttcac atttgcggct gagttcttag aaaagctgtt
     4921 tgtgagttcg ttggtgatcc ggtcatacaa atagttgagg gtggagatat attcattgtc
     4981 cagattcttg tcaagattgt acagccaggt cctagaaatc ctgctttctt cctcgttgtt
     5041 gtggaccacc cagtgcctca ggccagagag cagggcgagg atatcataac attcattccc
     5101 gtaattaatg atgtagttgc ggccctcttt gctgaagaag gcctggccaa aatagcccag
     5161 ccttgggttg tccaggaagt tgtcaaattc atcatactga gccttgatgg cattgatcaa
     5221 tttgtcatta ttgttgaagg cagctctgtg gtgctcgggg tctttaaatt catcgtaggt
     5281 atacactgtg gaaaacttcc caaacccaat aatatccttg tccaagcctg agatgttatt
     5341 aacagcatat gcagcatttg tgatgtactc agccaggatc ttttcaatat caagaatatt
     5401 atgaatcacc tgaatgcaga tattgtcatt tccatctgca gactccccga aataccgctt
     5461 ctccaaagtc tccttcagcc ccagcatgtc ctgctgcaca ggtcctgtgt agagaggatt
     5521 gttagcaacc actgcatatc ccttggggtg gctgaacttg gcgttcccga ttttgtaccc
     5581 agcattcttg tcagccattt ctgcactgaa agcttctcct tcattcacag agcggatgct
     5641 gtccccttcc acgatcttct ccagccgggc atcactcccc tctgcaaagg tggtcatgta
     5701 caccttgctg ccgctcacca gagtgctctt cacgcccatc cctttggcaa aggatttctt
     5761 cttctcaatc atggtcttgt cgacctgcag tttggattat tctgcgggca gaaaatagag
     5821 atgtggaaaa ttagtacgaa atcaaatgag tttcgttgaa attacaaaac tattgaaact
     5881 aacttcctgg ctggggaata aaaatgggaa acttatttat cgacgccaac tttgttgaga
     5941 aacccctatt aaccctctac gaatattgga acaaaggaaa gcgaagaaac agggaacaaa
     6001 gggtagttga gaaacctttg ttccgttgct tcgtcatcgt tttcgtgttg cgagtgtgtg
     6061 catgtatata tacacagctg aaatgcatgc atacacatta ttttgtgtgt atatggtgac
     6121 gtcacaacta caagcaataa gaaattttca gacgtggctt tgtttaagca acctactcta
     6181 tttcagctaa aaataagtgg atttcgttgg taaaatactt caattaacta aacaactaac
     6241 taataacatg cacacaaatg ctcgatcgag tgcgttcgtg atttctcgaa ttttcaaatg
     6301 cgtcactgcg aatttcacaa tttgccaata aatcttggcg aaaatcaaca cgcaagtttt
     6361 atttatagat ttgtttgcgt tttgatgcca attcattggg aaaacaagat gcgtggctgc
     6421 caatttctta ttttgtaatt acgtagagcg ttgaataaaa aaaaatggcc gaacaaagac
     6481 cttgaaatgc agtttttctt gaaattactc aacgtcttgt tgctcttatt actaattggt
     6541 aacagcgagt taaaaactta cgtttcttgt gactttcgag aatgttcttt taattgtact
     6601 ttaatcacca acaattaagt ataaattttt cgctgattgc gctttacttt ctacttgtac
     6661 ttgctgctgc aaatgtcaat tggttttgaa ggcgaccgtt cgcgaacgct gtttatatac
     6721 cttcggtgtc cgttgaaaat cactaaaaaa taccgtagtg ttcgtaacac tttagtacag
     6781 agaaaaaaaa ttgtgccgaa atgtttttga tacgtacgaa taccttgtat taaaattttt
     6841 tatgatttct gtgtatcact ttttttttgt gtttttcgtt taaactcacc acagtacaaa
     6901 acaataaaat atttttaaga caatttcaaa ttgagacctt tctcgtactg acttgaccgg
     6961 ctgaatgagg atttctacct agacgaccta cttcttacca tgacattgaa tgcaatgcca
     7021 cctttgatct aaacttacaa aagtccaagg cttgttagga ttggtgtttt ggaatttaag
     7081 acccttgtta gtttgctttt gaaatagcac tgtcttctct accggctata attttgaaac
     7141 tcgcagcttg actggaaatt taaaaagtaa ttctgtgtag gtaaagggtg ttttaaaagt
     7201 gtgatgtgtt gagcgttgcg gcaacgactg ctatttatgt atatattttc aaaacttatt
     7261 gtttttgaag tgttttaaat ggagctatct ggcaacgctg cgcataatct tacacaagct
     7321 agcttttctt aatccatttt taagtgaaat ttgtttatac tctttcggca aataattgtt
     7381 aaatcgcttt aagtgggctt acatctggat aagtaatgaa aacctgcata ttataatatt
     7441 aaaacatata atccactgtg ctttccccgt gtgtggccat atacctaaaa aagtttattt
     7501 tcgcagagcc ccgcacggtc acactacggt tcggcgattt tcgattttgg acagtactga
     7561 ttgcaagcgc accgaaagca aaatggagct ggagattttg aacgcgaaga acagcaagcc
     7621 gtacggcaag gtgaaggtgc cctccggcgc cacgcccatc ggcgatctgc gcgccctaat
     7681 tcacaagacc ctgaagcaga ccccacacgc gaatcgccag tcgcttcgtc tggaactgaa
     7741 gggcaaaagc ctgaaagata cggacacatt ggaatctctg tcgctgcgtt ccggcgacaa
     7801 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     7861 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     7921 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     7981 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggc
//
