LOCUS       sequence-398022-        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"
     primer_bind     complement(35..52)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(206..794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(286..305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(965..1756,1757..1825))
                     /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     1588..1607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1826..1930)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2076..2095
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(2189..2588)
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     polyA_signal    2671..2800
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     CDS             complement(2818..2835)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2851..2877)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(6196..6219)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(8268..8284)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8268..8284)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8281..8303)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8292..8308
                     /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(8316..8322,8323..8340,8341..8346))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8361..8382
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
       61 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      841 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      901 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
      961 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1021 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1081 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1141 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1201 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1261 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1321 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1381 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1441 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1501 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1561 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1621 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1681 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1741 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1801 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     1861 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1921 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     1981 tgacacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     2041 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     2101 agtgcaccat atgaaaaaaa gttgtaatag cccctcaaaa ctggaccttc cacaactgag
     2161 acgtacaact acaaatacaa ccgtctccga agtattgagg aaaacatacc tatataaatg
     2221 atcaacatca ggaaagagca gttgagaatt ataagaattg gcaaatggtc ttaagaaccc
     2281 tctgcttaag attttcaaaa tttcctttag aatcaaagtg tcctattgtt cgtttgttga
     2341 aaacacagtt cgatctattg actataataa attgatagtt ttaaatatag aggcacgact
     2401 aagagagttt ggttttgttt tggttaggga ccaaaaaaag tatacataac gaataaaaaa
     2461 ggatttaagt gcaaatggta aaaaagtgtc gagtttttct tgagttgatt gtgctgtaat
     2521 gagactctgc attcggcatt tgactcggct tttcctactc gtgccgtatt tcaggctgca
     2581 agtcgaacca tatggctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2641 gtattaccgc ctttgagtga gcatcgatcc cacgcgcttg aaaggagtgt gtaaatggac
     2701 aatgttgtta caataaaagt tgaaattatt ttataaaatt tattaaaaac atatttaaac
     2761 ataacatatt gtattaaaac atgacaatac aaactaagat ttagtcagat aaactcaatg
     2821 gtgatggtga tgatgaccgg tacgcgttca ggcgtagtca ggcacgtcgt aggggtagcc
     2881 acttcccttc atgatggcgt actccccaaa ggccttcttg atgctcatgg cgatctcagg
     2941 cagggccttg atttccacca cgcctttgtc ggggtaattg tttgcatcga aggcgttccg
     3001 gatcttccgc aggatgtcgc tctgctcttt gttgatgttc ttgttgttca gcaggatttt
     3061 caggatgctc ttgaagtcca ccttctcttc ccggtccact ctggcagaca gctcggggta
     3121 tgtgtcgaag gcccactttt ccaggttgaa cacgatggag ctgatctcgg gcctgcactg
     3181 gtcgtatttg ttgaactctt ccatgatatc ctctttggac acgatgtcgc tgcccacgag
     3241 ttccagcagg ttgccgatcc tcttgtcgct agccagcaca aagaagtcgc cgtagttctt
     3301 cagcttcatg ccctcgctgg tgatggtgta cttcttgccg cctttctcga aggtgaagct
     3361 catgggcatg atctcgctca ggattccctt ctcggcgtcg ggcatgattt ctttcagttt
     3421 gaacctctcg ccgtcgaaat cggccagttc ggtcagggtc tttttggcca gcagaaacag
     3481 cagggcatcc tgcactctgt agcgccggat cactttctcg cttttctggt actcgttccg
     3541 gctgttgctc agccgcttat ccaggattgt ctcgatctct tcgctgctgg cgtttctcat
     3601 ctgccggttg ctgcggatct tgttgctggc ctgctttctg taccgctctg ttctggaggc
     3661 ccgctctttc cagaggcctt ctctctcttc cacgctggtg aagcagtgct gcagggagcc
     3721 ctttctgtcg tactcgccct taagcatgtc catgtaccgg tagttgcggt tccactggta
     3781 gaaggtctgg aagtcgtcgt ccagcactct cttcatgtac tcggcgatca gataggtcac
     3841 gttggcattg ttgaagtcga tgccttccat ctgtggcagg gacttcaggt gggacttgat
     3901 ctcattgtcg aacatctgtc tgggcagttc cacgggcaga tcctcgctgt agattctgcc
     3961 cagggtcttc atggcgggtg ttttccactt gttctggtcc cgccggatga agggcacatc
     4021 cactctgttg cctttcttga tctcgttgga caggccggtc aggtagaact tccgctcgat
     4081 caggtagcgc tcgtagaact cgacggcatt ggcggggatg ctgcgggcga acaccttgta
     4141 cagaaatgga tgaggctctg ttgtgccctt gccgatcagc cgggccttct cgaacatcag
     4201 cttgaactgc tgcttggcct cgtaatcgtc gccgctatcg tacacggcaa tggcgctctg
     4261 catgatccgg tagttcaggc cggtgatctt gttctcgcca tcgttcacgc tgggctgaaa
     4321 cagcacgatg tccttggcca ggaagtcggc cagcttgcct gtggagatct gcttgaagcc
     4381 tctctttccc atcttgttgt cggcgctccg aatggacttc cggtcgtcct tgaatctctt
     4441 gattctccgc tcggtgtcgg tcagcatgtc gtccacggtc agtctgatga aggcgtccac
     4501 atccttgccg tgggcattgc cgctgatcag atccaggatc ttctgaggca ggtcgctctc
     4561 ggcgattccg aagctggcga tattctcggc ggtcacttct tctttctgca tggcctggaa
     4621 cagtctcttg taccggttgt gcacgtccac gatcagcttc tcggttttct tgctgccgaa
     4681 cagaaacatg tggaaggcca tggctggaat ttccagggtg ctcatccggc agctggggat
     4741 tgtcttgacc acgtatctat catcctcgat cacgggcagc agtggggcgc tgtcctcttt
     4801 gtcgttgata aacatctcga ccttgttgtt ttccaggatg tagtgtgtgt aggtgtccac
     4861 gatgtaggga tagttggcag gattggcgtc gtcccgcttc atgttctcga agtctctgat
     4921 ccggatgccg ctgttgccga aggtgccgtt ctcttgcttc cgcattgtct cggcctcttc
     4981 cagtctgccg aagccgttca ggggctgctc gatcactctg actctggtct ggccgtcgat
     5041 gcaggtcttg tcggccttca gcaggtatct cagcttgccc atgttcacgt ggaacctgat
     5101 gtggtcgaac agcttgccgt aatcgatata ctgcagcagc agaggcacga atctgtcgct
     5161 gctccgcttc atcagcactt cattgtggtc gtcgctgatg attctgaacc gggactgctt
     5221 ctcggcagac agtgttgtga acagctcgtc ggggcaccgc ttcacttcgt tgagcatatc
     5281 catggccacg ctcttgttgg acttctcgct gtggatccgg tccttgggca gcttgatgct
     5341 gttgatgccg aaggatctga tgatgatccg ccgttcctcg ctctgggcat tgtagctgga
     5401 gaagatgggc agcctgctca gaaagatgtt gatgtactgc ttgtccagga acaggcagat
     5461 cagcagggcg attcccactc cgctcaggtg cagcttcttc tgtgtgtcgc cgttgtagtc
     5521 ctgcaggctc aggaagaatc cggtattcac ttgggacttt ttcttgccgt aggcgtcctt
     5581 cacgaacttg aaccgcttgt cctggatgaa ggccaggtcc tctgtcttgt agccgtatct
     5641 ctcgttcatg ttccgcaggg ccactgtgta gtagttgttg atcatgccgc tcagaggttg
     5701 ctctgtgctg gtcaggaact cgcagccgtc gttcagcttt tcctcgtagg tcttgtatgc
     5761 gttggtcagg tccctgtaca tcttcagcac gccgaaggcg cgcttcagca cctcgaagat
     5821 gtcgttgctg ttcacttcca cgcggttctg cttgtacttg ccgttgctgt actctctctg
     5881 gttctcggcc atgatcttca ggaatgggaa gtagctctgc agccgctcga tgatgaacat
     5941 ggttttctcg ggctgcttgt cgtagccgtt cttggcgttg tacaggtggc tcatcacggg
     6001 gtgaaaccac agattctcgt tgttctcgtt ctgctcgccc tcaatatcgg ccaccttctg
     6061 gatgtggtcc agcacggtct gagcgttcag catggccatc acgctgtagg tgccaaagta
     6121 cttcttctgg ttttccacca gagcggggat gttagatcca cctttaatta acccggggag
     6181 ctcggtaccg ttaaccttat cgtcatcgtc tttgtaatcc atggtcttgt cgacctgcag
     6241 tttggattat tctgcgggca gaaaatagag atgtggaaaa ttagtacgaa atcaaatgag
     6301 tttcgttgaa attacaaaac tattgaaact aacttcctgg ctggggaata aaaatgggaa
     6361 acttatttat cgacgccaac tttgttgaga aacccctatt aaccctctac gaatattgga
     6421 acaaaggaaa gcgaagaaac agggaacaaa gggtagttga gaaacctttg ttccgttgct
     6481 tcgtcatcgt tttcgtgttg cgagtgtgtg catgtatata tacacagctg aaatgcatgc
     6541 atacacatta ttttgtgtgt atatggtgac gtcacaacta caagcaataa gaaattttca
     6601 gacgtggctt tgtttaagca acctactcta tttcagctaa aaataagtgg atttcgttgg
     6661 taaaatactt caattaacta aacaactaac taataacatg cacacaaatg ctcgatcgag
     6721 tgcgttcgtg atttctcgaa ttttcaaatg cgtcactgcg aatttcacaa tttgccaata
     6781 aatcttggcg aaaatcaaca cgcaagtttt atttatagat ttgtttgcgt tttgatgcca
     6841 attcattggg aaaacaagat gcgtggctgc caatttctta ttttgtaatt acgtagagcg
     6901 ttgaataaaa aaaaatggcc gaacaaagac cttgaaatgc agtttttctt gaaattactc
     6961 aacgtcttgt tgctcttatt actaattggt aacagcgagt taaaaactta cgtttcttgt
     7021 gactttcgag aatgttcttt taattgtact ttaatcacca acaattaagt ataaattttt
     7081 cgctgattgc gctttacttt ctacttgtac ttgctgctgc aaatgtcaat tggttttgaa
     7141 ggcgaccgtt cgcgaacgct gtttatatac cttcggtgtc cgttgaaaat cactaaaaaa
     7201 taccgtagtg ttcgtaacac tttagtacag agaaaaaaaa ttgtgccgaa atgtttttga
     7261 tacgtacgaa taccttgtat taaaattttt tatgatttct gtgtatcact ttttttttgt
     7321 gtttttcgtt taaactcacc acagtacaaa acaataaaat atttttaaga caatttcaaa
     7381 ttgagacctt tctcgtactg acttgaccgg ctgaatgagg atttctacct agacgaccta
     7441 cttcttacca tgacattgaa tgcaatgcca cctttgatct aaacttacaa aagtccaagg
     7501 cttgttagga ttggtgtttt ggaatttaag acccttgtta gtttgctttt gaaatagcac
     7561 tgtcttctct accggctata attttgaaac tcgcagcttg actggaaatt taaaaagtaa
     7621 ttctgtgtag gtaaagggtg ttttaaaagt gtgatgtgtt gagcgttgcg gcaacgactg
     7681 ctatttatgt atatattttc aaaacttatt gtttttgaag tgttttaaat ggagctatct
     7741 ggcaacgctg cgcataatct tacacaagct agcttttctt aatccatttt taagtgaaat
     7801 ttgtttatac tctttcggca aataattgtt aaatcgcttt aagtgggctt acatctggat
     7861 aagtaatgaa aacctgcata ttataatatt aaaacatata atccactgtg ctttccccgt
     7921 gtgtggccat atacctaaaa aagtttattt tcgcagagcc ccgcacggtc acactacggt
     7981 tcggcgattt tcgattttgg acagtactga ttgcaagcgc accgaaagca aaatggagct
     8041 ggagattttg aacgcgaaga acagcaagcc gtacggcaag gtgaaggtgc cctccggcgc
     8101 cacgcccatc ggcgatctgc gcgccctaat tcacaagacc ctgaagcaga ccccacacgc
     8161 gaatcgccag tcgcttcgtc tggaactgaa gggcaaaagc ctgaaagata cggacacatt
     8221 ggaatctctg tcgctgcgtt ccggcgacaa agcttggcgt aatcatggtc atagctgttt
     8281 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     8341 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     8401 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     8461 ggga
//
