LOCUS       sequence-349455-        8464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 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 PspCas13b + PspCas13b guide RNA
FEATURES             Location/Qualifiers
     source          1..8464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        103..207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(208..276,277..1068)
                     /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(426..445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1239..1827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1728..1747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1981..1998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2115..2136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2151..2156,2157..2174,2175..2181)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2189..2205
                     /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     2194..2216
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2213..2229
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2213..2229
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4278..4301
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7620..7646
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7662..7679
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    7697..7826
                     /gene="immediate-early gene 2 from the Orgyia pseudotsugata
                     multicapsid nucleopolyhedrovirus"
                     /label=OpIE-2 poly(A) signal
                     /note="baculovirus polyadenylation signal"
     promoter        7909..8308
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     primer_bind     complement(8402..8421)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtgt catgataata
       61 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
      121 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
      181 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
      241 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
      301 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
      361 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
      421 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
      481 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
      541 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
      601 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      661 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
      721 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
      781 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
      841 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
      901 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
      961 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1021 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1081 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1141 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1201 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1261 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1321 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1381 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1441 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1501 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1561 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1621 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1681 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1741 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1801 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     1861 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     1921 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     1981 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     2041 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     2101 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     2161 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     2221 agctatgacc atgattacgc caagctttgt cgccggaacg cagcgacaga gattccaatg
     2281 tgtccgtatc tttcaggctt ttgcccttca gttccagacg aagcgactgg cgattcgcgt
     2341 gtggggtctg cttcagggtc ttgtgaatta gggcgcgcag atcgccgatg ggcgtggcgc
     2401 cggagggcac cttcaccttg ccgtacggct tgctgttctt cgcgttcaaa atctccagct
     2461 ccattttgct ttcggtgcgc ttgcaatcag tactgtccaa aatcgaaaat cgccgaaccg
     2521 tagtgtgacc gtgcggggct ctgcgaaaat aaactttttt aggtatatgg ccacacacgg
     2581 ggaaagcaca gtggattata tgttttaata ttataatatg caggttttca ttacttatcc
     2641 agatgtaagc ccacttaaag cgatttaaca attatttgcc gaaagagtat aaacaaattt
     2701 cacttaaaaa tggattaaga aaagctagct tgtgtaagat tatgcgcagc gttgccagat
     2761 agctccattt aaaacacttc aaaaacaata agttttgaaa atatatacat aaatagcagt
     2821 cgttgccgca acgctcaaca catcacactt ttaaaacacc ctttacctac acagaattac
     2881 tttttaaatt tccagtcaag ctgcgagttt caaaattata gccggtagag aagacagtgc
     2941 tatttcaaaa gcaaactaac aagggtctta aattccaaaa caccaatcct aacaagcctt
     3001 ggacttttgt aagtttagat caaaggtggc attgcattca atgtcatggt aagaagtagg
     3061 tcgtctaggt agaaatcctc attcagccgg tcaagtcagt acgagaaagg tctcaatttg
     3121 aaattgtctt aaaaatattt tattgttttg tactgtggtg agtttaaacg aaaaacacaa
     3181 aaaaaaagtg atacacagaa atcataaaaa attttaatac aaggtattcg tacgtatcaa
     3241 aaacatttcg gcacaatttt ttttctctgt actaaagtgt tacgaacact acggtatttt
     3301 ttagtgattt tcaacggaca ccgaaggtat ataaacagcg ttcgcgaacg gtcgccttca
     3361 aaaccaattg acatttgcag cagcaagtac aagtagaaag taaagcgcaa tcagcgaaaa
     3421 atttatactt aattgttggt gattaaagta caattaaaag aacattctcg aaagtcacaa
     3481 gaaacgtaag tttttaactc gctgttacca attagtaata agagcaacaa gacgttgagt
     3541 aatttcaaga aaaactgcat ttcaaggtct ttgttcggcc attttttttt attcaacgct
     3601 ctacgtaatt acaaaataag aaattggcag ccacgcatct tgttttccca atgaattggc
     3661 atcaaaacgc aaacaaatct ataaataaaa cttgcgtgtt gattttcgcc aagatttatt
     3721 ggcaaattgt gaaattcgca gtgacgcatt tgaaaattcg agaaatcacg aacgcactcg
     3781 atcgagcatt tgtgtgcatg ttattagtta gttgtttagt taattgaagt attttaccaa
     3841 cgaaatccac ttatttttag ctgaaataga gtaggttgct taaacaaagc cacgtctgaa
     3901 aatttcttat tgcttgtagt tgtgacgtca ccatatacac acaaaataat gtgtatgcat
     3961 gcatttcagc tgtgtatata tacatgcaca cactcgcaac acgaaaacga tgacgaagca
     4021 acggaacaaa ggtttctcaa ctaccctttg ttccctgttt cttcgctttc ctttgttcca
     4081 atattcgtag agggttaata ggggtttctc aacaaagttg gcgtcgataa ataagtttcc
     4141 catttttatt ccccagccag gaagttagtt tcaatagttt tgtaatttca acgaaactca
     4201 tttgatttcg tactaatttt ccacatctct attttctgcc cgcagaataa tccaaactgc
     4261 aggtcgacaa gaccatggat tacaaagacg atgacgataa ggttaacggt accgagctcc
     4321 ccgggttaat taaaggtgga tctaacatcc ccgctctggt ggaaaaccag aagaagtact
     4381 ttggcaccta cagcgtgatg gccatgctga acgctcagac cgtgctggac cacatccaga
     4441 aggtggccga tattgagggc gagcagaacg agaacaacga gaatctgtgg tttcaccccg
     4501 tgatgagcca cctgtacaac gccaagaacg gctacgacaa gcagcccgag aaaaccatgt
     4561 tcatcatcga gcggctgcag agctacttcc cattcctgaa gatcatggcc aagaaccaga
     4621 gagagtacag caacggcaag tacaagcaga accgcgtgga agtgaacagc aacgacatct
     4681 tcgaggtgct gaagcgcgcc ttcggcgtgc tgaagatgta cagggacctg accaaccact
     4741 acaagaccta cgaggaaaag ctgaacgacg gctgcgagtt cctgaccagc acagagcaac
     4801 ctctgagcgg catgatcaac aactactaca cagtggccct gcggaacatg aacgagagat
     4861 acggctacaa gacagaggac ctggccttca tccaggacaa gcggttcaag ttcgtgaagg
     4921 acgcctacgg caagaaaaag tcccaagtga ataccggatt cttcctgagc ctgcaggact
     4981 acaacggcga cacacagaag aagctgcacc tgagcggagt gggaatcgcc ctgctgatct
     5041 gcctgttcct ggacaagcag tacatcagca tctttctgag caggctgccc atcttctcca
     5101 gctacaatgc ccagagcgag gaacggcgga tcatcatcag atccttcggc atcaacagca
     5161 tcaagctgcc caaggaccgg atccacagcg agaagtccaa caagagcgtg gccatggata
     5221 tgctcaacga agtgaagcgg tgccccgacg agctgttcac aacactgtct gccgagaagc
     5281 agtcccggtt cagaatcatc agcgacgacc acaatgaagt gctgatgaag cggagcagcg
     5341 acagattcgt gcctctgctg ctgcagtata tcgattacgg caagctgttc gaccacatca
     5401 ggttccacgt gaacatgggc aagctgagat acctgctgaa ggccgacaag acctgcatcg
     5461 acggccagac cagagtcaga gtgatcgagc agcccctgaa cggcttcggc agactggaag
     5521 aggccgagac aatgcggaag caagagaacg gcaccttcgg caacagcggc atccggatca
     5581 gagacttcga gaacatgaag cgggacgacg ccaatcctgc caactatccc tacatcgtgg
     5641 acacctacac acactacatc ctggaaaaca acaaggtcga gatgtttatc aacgacaaag
     5701 aggacagcgc cccactgctg cccgtgatcg aggatgatag atacgtggtc aagacaatcc
     5761 ccagctgccg gatgagcacc ctggaaattc cagccatggc cttccacatg tttctgttcg
     5821 gcagcaagaa aaccgagaag ctgatcgtgg acgtgcacaa ccggtacaag agactgttcc
     5881 aggccatgca gaaagaagaa gtgaccgccg agaatatcgc cagcttcgga atcgccgaga
     5941 gcgacctgcc tcagaagatc ctggatctga tcagcggcaa tgcccacggc aaggatgtgg
     6001 acgccttcat cagactgacc gtggacgaca tgctgaccga caccgagcgg agaatcaaga
     6061 gattcaagga cgaccggaag tccattcgga gcgccgacaa caagatggga aagagaggct
     6121 tcaagcagat ctccacaggc aagctggccg acttcctggc caaggacatc gtgctgtttc
     6181 agcccagcgt gaacgatggc gagaacaaga tcaccggcct gaactaccgg atcatgcaga
     6241 gcgccattgc cgtgtacgat agcggcgacg attacgaggc caagcagcag ttcaagctga
     6301 tgttcgagaa ggcccggctg atcggcaagg gcacaacaga gcctcatcca tttctgtaca
     6361 aggtgttcgc ccgcagcatc cccgccaatg ccgtcgagtt ctacgagcgc tacctgatcg
     6421 agcggaagtt ctacctgacc ggcctgtcca acgagatcaa gaaaggcaac agagtggatg
     6481 tgcccttcat ccggcgggac cagaacaagt ggaaaacacc cgccatgaag accctgggca
     6541 gaatctacag cgaggatctg cccgtggaac tgcccagaca gatgttcgac aatgagatca
     6601 agtcccacct gaagtccctg ccacagatgg aaggcatcga cttcaacaat gccaacgtga
     6661 cctatctgat cgccgagtac atgaagagag tgctggacga cgacttccag accttctacc
     6721 agtggaaccg caactaccgg tacatggaca tgcttaaggg cgagtacgac agaaagggct
     6781 ccctgcagca ctgcttcacc agcgtggaag agagagaagg cctctggaaa gagcgggcct
     6841 ccagaacaga gcggtacaga aagcaggcca gcaacaagat ccgcagcaac cggcagatga
     6901 gaaacgccag cagcgaagag atcgagacaa tcctggataa gcggctgagc aacagccgga
     6961 acgagtacca gaaaagcgag aaagtgatcc ggcgctacag agtgcaggat gccctgctgt
     7021 ttctgctggc caaaaagacc ctgaccgaac tggccgattt cgacggcgag aggttcaaac
     7081 tgaaagaaat catgcccgac gccgagaagg gaatcctgag cgagatcatg cccatgagct
     7141 tcaccttcga gaaaggcggc aagaagtaca ccatcaccag cgagggcatg aagctaaaga
     7201 actacggcga cttctttgtg ctggctagcg acaagaggat cggcaacctg ctggaactcg
     7261 tgggcagcga catcgtgtcc aaagaggata tcatggaaga gttcaacaaa tacgaccagt
     7321 gcaggcccga gatcagctcc atcgtgttca acctggaaaa gtgggccttc gacacatacc
     7381 ccgagctgtc tgccagagtg gaccgggaag agaaggtgga cttcaagagc atcctgaaaa
     7441 tcctgctgaa caacaagaac atcaacaaag agcagagcga catcctgcgg aagatccgga
     7501 acgccttcga tcacaacaat taccccgaca aaggcgtggt ggaaatcaag gccctgcctg
     7561 agatcgccat gagcatcaag aaggcctttg gggagtacgc catcatgaag ggaagtggct
     7621 acccctacga cgtgcctgac tacgcctgaa cgcgtaccgg tcatcatcac catcaccatt
     7681 gagtttatct gactaaatct tagtttgtat tgtcatgttt taatacaata tgttatgttt
     7741 aaatatgttt ttaataaatt ttataaaata atttcaactt ttattgtaac aacattgtcc
     7801 atttacacac tcctttcaag cgcgtgggat cgatgctcac tcaaaggcgg taatacggtt
     7861 atccacagaa tcaggggata acgcaggaaa gaacatgtga gccatatggt tcgacttgca
     7921 gcctgaaata cggcacgagt aggaaaagcc gagtcaaatg ccgaatgcag agtctcatta
     7981 cagcacaatc aactcaagaa aaactcgaca cttttttacc atttgcactt aaatcctttt
     8041 ttattcgtta tgtatacttt ttttggtccc taaccaaaac aaaaccaaac tctcttagtc
     8101 gtgcctctat atttaaaact atcaatttat tatagtcaat agatcgaact gtgttttcaa
     8161 caaacgaaca ataggacact ttgattctaa aggaaatttt gaaaatctta agcagagggt
     8221 tcttaagacc atttgccaat tcttataatt ctcaactgct ctttcctgat gttgatcatt
     8281 tatataggta tgttttcctc aatacttcgg agacggttgt atttgtagtt gtacgtctca
     8341 gttgtggaag gtccagtttt gaggggctat tacaactttt tttcatatgg tgcactctca
     8401 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     8461 acgc
//
