LOCUS       sequence-346361-        8464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses catalytic dead PspCas13b and its associated guide RNA in 
            Drosophila cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8464)
  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 8464)
  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 dPspCas13b + PspCas13b guide RNA
FEATURES             Location/Qualifiers
     source          1..8464
                     /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             4231..4254
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7573..7599
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7615..7632
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7650..7779
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     promoter        7862..8261
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     primer_bind     complement(8355..8374)
                     /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 caagaccatg gattacaaag acgatgacga taaggttaac
     4261 ggtaccgagc tccccgggtt aattaaaggt ggatctaaca tccccgctct ggtggaaaac
     4321 cagaagaagt actttggcac ctacagcgtg atggccatgc tgaacgctca gaccgtgctg
     4381 gaccacatcc agaaggtggc cgatattgag ggcgagcaga acgagaacaa cgagaatctg
     4441 tggtttcacc ccgtgatgag ccacctgtac aacgccaaga acggctacga caagcagccc
     4501 gagaaaacca tgttcatcat cgagcggctg cagagctact tcccattcct gaagatcatg
     4561 gccgagaacc agagagagta cagcaacggc aagtacaagc agaaccgcgt ggaagtgaac
     4621 agcaacgaca tcttcgaggt gctgaagcgc gccttcggcg tgctgaagat gtacagggac
     4681 ctgaccaacg catacaagac ctacgaggaa aagctgaacg acggctgcga gttcctgacc
     4741 agcacagagc aacctctgag cggcatgatc aacaactact acacagtggc cctgcggaac
     4801 atgaacgaga gatacggcta caagacagag gacctggcct tcatccagga caagcggttc
     4861 aagttcgtga aggacgccta cggcaagaaa aagtcccaag tgaataccgg attcttcctg
     4921 agcctgcagg actacaacgg cgacacacag aagaagctgc acctgagcgg agtgggaatc
     4981 gccctgctga tctgcctgtt cctggacaag cagtacatca acatctttct gagcaggctg
     5041 cccatcttct ccagctacaa tgcccagagc gaggaacggc ggatcatcat cagatccttc
     5101 ggcatcaaca gcatcaagct gcccaaggac cggatccaca gcgagaagtc caacaagagc
     5161 gtggccatgg atatgctcaa cgaagtgaag cggtgccccg acgagctgtt cacaacactg
     5221 tctgccgaga agcagtcccg gttcagaatc atcagcgacg accacaatga agtgctgatg
     5281 aagcggagca gcgacagatt cgtgcctctg ctgctgcagt atatcgatta cggcaagctg
     5341 ttcgaccaca tcaggttcca cgtgaacatg ggcaagctga gatacctgct gaaggccgac
     5401 aagacctgca tcgacggcca gaccagagtc agagtgatcg agcagcccct gaacggcttc
     5461 ggcagactgg aagaggccga gacaatgcgg aagcaagaga acggcacctt cggcaacagc
     5521 ggcatccgga tcagagactt cgagaacatg aagcgggacg acgccaatcc tgccaactat
     5581 ccctacatcg tggacaccta cacacactac atcctggaaa acaacaaggt cgagatgttt
     5641 atcaacgaca aagaggacag cgccccactg ctgcccgtga tcgaggatga tagatacgtg
     5701 gtcaagacaa tccccagctg ccggatgagc accctggaaa ttccagccat ggccttccac
     5761 atgtttctgt tcggcagcaa gaaaaccgag aagctgatcg tggacgtgca caaccggtac
     5821 aagagactgt tccaggccat gcagaaagaa gaagtgaccg ccgagaatat cgccagcttc
     5881 ggaatcgccg agagcgacct gcctcagaag atcctggatc tgatcagcgg caatgcccac
     5941 ggcaaggatg tggacgcctt catcagactg accgtggacg acatgctgac cgacaccgag
     6001 cggagaatca agagattcaa ggacgaccgg aagtccattc ggagcgccga caacaagatg
     6061 ggaaagagag gcttcaagca gatctccaca ggcaagctgg ccgacttcct ggccaaggac
     6121 atcgtgctgt ttcagcccag cgtgaacgat ggcgagaaca agatcaccgg cctgaactac
     6181 cggatcatgc agagcgccat tgccgtgtac gatagcggcg acgattacga ggccaagcag
     6241 cagttcaagc tgatgttcga gaaggcccgg ctgatcggca agggcacaac agagcctcat
     6301 ccatttctgt acaaggtgtt cgcccgcagc atccccgcca atgccgtcga gttctacgag
     6361 cgctacctga tcgagcggaa gttctacctg accggcctgt ccaacgagat caagaaaggc
     6421 aacagagtgg atgtgccctt catccggcgg gaccagaaca agtggaaaac acccgccatg
     6481 aagaccctgg gcagaatcta cagcgaggat ctgcccgtgg aactgcccag acagatgttc
     6541 gacaatgaga tcaagtccca cctgaagtcc ctgccacaga tggaaggcat cgacttcaac
     6601 aatgccaacg tgacctatct gatcgccgag tacatgaaga gagtgctgga cgacgacttc
     6661 cagaccttct accagtggaa ccgcaactac cggtacatgg acatgcttaa gggcgagtac
     6721 gacagaaagg gctccctgca gcactgcttc accagcgtgg aagagagaga aggcctctgg
     6781 aaagagcggg cctccagaac agagcggtac agaaagcagg ccagcaacaa gatccgcagc
     6841 aaccggcaga tgagaaacgc cagcagcgaa gagatcgaga caatcctgga taagcggctg
     6901 agcaacagcc ggaacgagta ccagaaaagc gagaaagtga tccggcgcta cagagtgcag
     6961 gatgccctgc tgtttctgct ggccaaaaag accctgaccg aactggccga tttcgacggc
     7021 gagaggttca aactgaaaga aatcatgccc gacgccgaga agggaatcct gagcgagatc
     7081 atgcccatga gcttcacctt cgagaaaggc ggcaagaagt acaccatcac cagcgagggc
     7141 atgaagctga agaactacgg cgacttcttt gtgctggcta gcgacaagag gatcggcaac
     7201 ctgctggaac tcgtgggcag cgacatcgtg tccaaagagg atatcatgga agagttcaac
     7261 aaatacgacc agtgcaggcc cgagatcagc tccatcgtgt tcaacctgga aaagtgggcc
     7321 ttcgacacat accccgagct gtctgccaga gtggaccggg aagagaaggt ggacttcaag
     7381 agcatcctga aaatcctgct gaacaacaag aacatcaaca aagagcagag cgacatcctg
     7441 cggaagatcc ggaacgcctt cgatgcaaac aattaccccg acaaaggcgt ggtggaaatc
     7501 aaggccctgc ctgagatcgc catgagcatc aagaaggcct ttggggagta cgccatcatg
     7561 aagggaagtg gctaccccta cgacgtgcct gactacgcct gaacgcgtac cggtcatcat
     7621 caccatcacc attgagttta tctgactaaa tcttagtttg tattgtcatg ttttaataca
     7681 atatgttatg tttaaatatg tttttaataa attttataaa ataatttcaa cttttattgt
     7741 aacaacattg tccatttaca cactcctttc aagcgcgtgg gatcgatgct cactcaaagg
     7801 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagccatat
     7861 ggttcgactt gcagcctgaa atacggcacg agtaggaaaa gccgagtcaa atgccgaatg
     7921 cagagtctca ttacagcaca atcaactcaa gaaaaactcg acactttttt accatttgca
     7981 cttaaatcct tttttattcg ttatgtatac tttttttggt ccctaaccaa aacaaaacca
     8041 aactctctta gtcgtgcctc tatatttaaa actatcaatt tattatagtc aatagatcga
     8101 actgtgtttt caacaaacga acaataggac actttgattc taaaggaaat tttgaaaatc
     8161 ttaagcagag ggttcttaag accatttgcc aattcttata attctcaact gctctttcct
     8221 gatgttgatc atttatatag gtatgttttc ctcaatactt cggagacggt tgtatttgta
     8281 gttgtacgtc tcagttgtgg aaggtccagt tttgaggggc tattacaact ttttttcata
     8341 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     8401 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     8461 gctg
//
