LOCUS       sequence-465658-        8949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of catalytically-inactive PspCas13b in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8949)
  AUTHORS   Apostolopoulos A, Kawamoto N, Chow SYA, Tsuiji H, Ikeuchi Y, 
            Shichino Y, Iwasaki S
  TITLE     dCas13-mediated translational repression for accurate gene silencing
            in mammalian cells.
  JOURNAL   Nat Commun. 2024 Mar 11;15(1):2205. doi: 10.1038/s41467-024-46412-7.
  PUBMED    38467613
REFERENCE   2  (bases 1 to 8949)
  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: "Nat 
            Commun."; date: "2024-03-11"; volume: "15(1):2205. doi"; pages: " 
            10.1038/s41467-024-46412-7"
COMMENT     Sequence Label: pCAGEN-dPspCas13b-NES-mCherry
FEATURES             Location/Qualifiers
     source          1..8949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..660
                     /label=chicken beta-actin promoter
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1748..1813
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1823..1847
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5138..5164
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             5174..5884
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5319..5337)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5587..5606
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5970..5989)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6035..6090
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6089..6108)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6451..6467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6451..6467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6464..6486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6475..6491
                     /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(6499..6505,6506..6523,6524..6529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6544..6565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6624..6819
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6670..6805
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6825..6959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6862..6881)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6916..6935
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7027..7044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7198..7786)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7278..7297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7957..8748,8749..8817))
                     /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     8580..8599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8818..8922)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt cctcgaggat
     1741 caccatggac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     1801 tgacgatgac aagcaccggt atacaagttt gtacaaaaaa gcaggctccg cggccgctat
     1861 gaacatcccc gctctggtgg aaaaccagaa gaagtacttt ggcacctaca gcgtgatggc
     1921 catgctgaac gctcagaccg tgctggacca catccagaag gtggccgata ttgagggcga
     1981 gcagaacgag aacaacgaga atctgtggtt tcaccccgtg atgagccacc tgtacaacgc
     2041 caagaacggc tacgacaagc agcccgagaa aaccatgttc atcatcgagc ggctgcagag
     2101 ctacttccca ttcctgaaga tcatggccga gaaccagaga gagtacagca acggcaagta
     2161 caagcagaac cgcgtggaag tgaacagcaa cgacatcttc gaggtgctga agcgcgcctt
     2221 cggcgtgctg aagatgtaca gggacctgac caacgcatac aagacctacg aggaaaagct
     2281 gaacgacggc tgcgagttcc tgaccagcac agagcaacct ctgagcggca tgatcaacaa
     2341 ctactacaca gtggccctgc ggaacatgaa cgagagatac ggctacaaga cagaggacct
     2401 ggccttcatc caggacaagc ggttcaagtt cgtgaaggac gcctacggca agaaaaagtc
     2461 ccaagtgaat accggattct tcctgagcct gcaggactac aacggcgaca cacagaagaa
     2521 gctgcacctg agcggagtgg gaatcgccct gctgatctgc ctgttcctgg acaagcagta
     2581 catcaacatc tttctgagca ggctgcccat cttctccagc tacaatgccc agagcgagga
     2641 acggcggatc atcatcagat ccttcggcat caacagcatc aagctgccca aggaccggat
     2701 ccacagcgag aagtccaaca agagcgtggc catggatatg ctcaacgaag tgaagcggtg
     2761 ccccgacgag ctgttcacaa cactgtctgc cgagaagcag tcccggttca gaatcatcag
     2821 cgacgaccac aatgaagtgc tgatgaagcg gagcagcgac agattcgtgc ctctgctgct
     2881 gcagtatatc gattacggca agctgttcga ccacatcagg ttccacgtga acatgggcaa
     2941 gctgagatac ctgctgaagg ccgacaagac ctgcatcgac ggccagacca gagtcagagt
     3001 gatcgagcag cccctgaacg gcttcggcag actggaagag gccgagacaa tgcggaagca
     3061 agagaacggc accttcggca acagcggcat ccggatcaga gacttcgaga acatgaagcg
     3121 ggacgacgcc aatcctgcca actatcccta catcgtggac acctacacac actacatcct
     3181 ggaaaacaac aaggtcgaga tgtttatcaa cgacaaagag gacagcgccc cactgctgcc
     3241 cgtgatcgag gatgatagat acgtggtcaa gacaatcccc agctgccgga tgagcaccct
     3301 ggaaattcca gccatggcct tccacatgtt tctgttcggc agcaagaaaa ccgagaagct
     3361 gatcgtggac gtgcacaacc ggtacaagag actgttccag gccatgcaga aagaagaagt
     3421 gaccgccgag aatatcgcca gcttcggaat cgccgagagc gacctgcctc agaagatcct
     3481 ggatctgatc agcggcaatg cccacggcaa ggatgtggac gccttcatca gactgaccgt
     3541 ggacgacatg ctgaccgaca ccgagcggag aatcaagaga ttcaaggacg accggaagtc
     3601 cattcggagc gccgacaaca agatgggaaa gagaggcttc aagcagatct ccacaggcaa
     3661 gctggccgac ttcctggcca aggacatcgt gctgtttcag cccagcgtga acgatggcga
     3721 gaacaagatc accggcctga actaccggat catgcagagc gccattgccg tgtacgatag
     3781 cggcgacgat tacgaggcca agcagcagtt caagctgatg ttcgagaagg cccggctgat
     3841 cggcaagggc acaacagagc ctcatccatt tctgtacaag gtgttcgccc gcagcatccc
     3901 cgccaatgcc gtcgagttct acgagcgcta cctgatcgag cggaagttct acctgaccgg
     3961 cctgtccaac gagatcaaga aaggcaacag agtggatgtg cccttcatcc ggcgggacca
     4021 gaacaagtgg aaaacacccg ccatgaagac cctgggcaga atctacagcg aggatctgcc
     4081 cgtggaactg cccagacaga tgttcgacaa tgagatcaag tcccacctga agtccctgcc
     4141 acagatggaa ggcatcgact tcaacaatgc caacgtgacc tatctgatcg ccgagtacat
     4201 gaagagagtg ctggacgacg acttccagac cttctaccag tggaaccgca actaccggta
     4261 catggacatg cttaagggcg agtacgacag aaagggctcc ctgcagcact gcttcaccag
     4321 cgtggaagag agagaaggcc tctggaaaga gcgggcctcc agaacagagc ggtacagaaa
     4381 gcaggccagc aacaagatcc gcagcaaccg gcagatgaga aacgccagca gcgaagagat
     4441 cgagacaatc ctggataagc ggctgagcaa cagccggaac gagtaccaga aaagcgagaa
     4501 agtgatccgg cgctacagag tgcaggatgc cctgctgttt ctgctggcca aaaagaccct
     4561 gaccgaactg gccgatttcg acggcgagag gttcaaactg aaagaaatca tgcccgacgc
     4621 cgagaaggga atcctgagcg agatcatgcc catgagcttc accttcgaga aaggcggcaa
     4681 gaagtacacc atcaccagcg agggcatgaa gctgaagaac tacggcgact tctttgtgct
     4741 ggctagcgac aagaggatcg gcaacctgct ggaactcgtg ggcagcgaca tcgtgtccaa
     4801 agaggatatc atggaagagt tcaacaaata cgaccagtgc aggcccgaga tcagctccat
     4861 cgtgttcaac ctggaaaagt gggccttcga cacatacccc gagctgtctg ccagagtgga
     4921 ccgggaagag aaggtggact tcaagagcat cctgaaaatc ctgctgaaca acaagaacat
     4981 caacaaagag cagagcgaca tcctgcggaa gatccggaac gccttcgatg caaacaatta
     5041 ccccgacaaa ggcgtggtgg aaatcaaggc cctgcctgag atcgccatga gcatcaagaa
     5101 ggcctttggg gagtacgcca tcatgaaggg atccggactg cctccacttg aaagactgac
     5161 actggcggcc gctatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     5221 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     5281 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     5341 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     5401 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     5461 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     5521 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     5581 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     5641 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     5701 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     5761 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     5821 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     5881 gtaagcggcc gcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     5941 aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac
     6001 atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca
     6061 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     6121 ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca tatgctggct
     6181 gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc
     6241 cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt
     6301 gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact agccagattt
     6361 ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg gagatccctc
     6421 gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6481 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6541 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6601 aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta
     6661 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     6721 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     6781 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag
     6841 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     6901 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc
     6961 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     7021 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     7081 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     7141 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     7201 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     7261 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     7321 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     7381 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     7441 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     7501 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     7561 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7621 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7681 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7741 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7801 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7861 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7921 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7981 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     8041 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     8101 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     8161 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     8221 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     8281 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     8341 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     8401 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     8461 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     8521 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8581 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8641 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8701 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8761 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     8821 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8881 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8941 gccacctgg
//
