LOCUS       sequence-465660-        8950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of PspCas13b in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8950)
  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 8950)
  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-PspCas13b-NES-mCherry
FEATURES             Location/Qualifiers
     source          1..8950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1749..1814
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    1824..1848
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5139..5165
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             5175..5885
                     /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(5320..5338)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5588..5607
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5971..5990)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6036..6091
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6090..6109)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6452..6468)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6452..6468)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6465..6487)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6476..6492
                     /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(6500..6506,6507..6524,6525..6530))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6545..6566
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6625..6820
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6671..6806
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6826..6960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6863..6882)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6917..6936
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7028..7045)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7199..7787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7279..7298)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7958..8749,8750..8818))
                     /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     8581..8600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8819..8923)
                     /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 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagga
     1741 tcaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc gattacaagg
     1801 atgacgatga caagcaccgg tatacaagtt tgtacaaaaa agcaggctcc gcggccgcta
     1861 tgaacatccc cgctctggtg gaaaaccaga agaagtactt tggcacctac agcgtgatgg
     1921 ccatgctgaa cgctcagacc gtgctggacc acatccagaa ggtggccgat attgagggcg
     1981 agcagaacga gaacaacgag aatctgtggt ttcaccccgt gatgagccac ctgtacaacg
     2041 ccaagaacgg ctacgacaag cagcccgaga aaaccatgtt catcatcgag cggctgcaga
     2101 gctacttccc attcctgaag atcatggccg agaaccagag agagtacagc aacggcaagt
     2161 acaagcagaa ccgcgtggaa gtgaacagca acgacatctt cgaggtgctg aagcgcgcct
     2221 tcggcgtgct gaagatgtac agggacctga ccaaccacta caagacctac gaggaaaagc
     2281 tgaacgacgg ctgcgagttc ctgaccagca cagagcaacc tctgagcggc atgatcaaca
     2341 actactacac agtggccctg cggaacatga acgagagata cggctacaag acagaggacc
     2401 tggccttcat ccaggacaag cggttcaagt tcgtgaagga cgcctacggc aagaaaaagt
     2461 cccaagtgaa taccggattc ttcctgagcc tgcaggacta caacggcgac acacagaaga
     2521 agctgcacct gagcggagtg ggaatcgccc tgctgatctg cctgttcctg gacaagcagt
     2581 acatcaacat ctttctgagc aggctgccca tcttctccag ctacaatgcc cagagcgagg
     2641 aacggcggat catcatcaga tccttcggca tcaacagcat caagctgccc aaggaccgga
     2701 tccacagcga gaagtccaac aagagcgtgg ccatggatat gctcaacgaa gtgaagcggt
     2761 gccccgacga gctgttcaca acactgtctg ccgagaagca gtcccggttc agaatcatca
     2821 gcgacgacca caatgaagtg ctgatgaagc ggagcagcga cagattcgtg cctctgctgc
     2881 tgcagtatat cgattacggc aagctgttcg accacatcag gttccacgtg aacatgggca
     2941 agctgagata cctgctgaag gccgacaaga cctgcatcga cggccagacc agagtcagag
     3001 tgatcgagca gcccctgaac ggcttcggca gactggaaga ggccgagaca atgcggaagc
     3061 aagagaacgg caccttcggc aacagcggca tccggatcag agacttcgag aacatgaagc
     3121 gggacgacgc caatcctgcc aactatccct acatcgtgga cacctacaca cactacatcc
     3181 tggaaaacaa caaggtcgag atgtttatca acgacaaaga ggacagcgcc ccactgctgc
     3241 ccgtgatcga ggatgataga tacgtggtca agacaatccc cagctgccgg atgagcaccc
     3301 tggaaattcc agccatggcc ttccacatgt ttctgttcgg cagcaagaaa accgagaagc
     3361 tgatcgtgga cgtgcacaac cggtacaaga gactgttcca ggccatgcag aaagaagaag
     3421 tgaccgccga gaatatcgcc agcttcggaa tcgccgagag cgacctgcct cagaagatcc
     3481 tggatctgat cagcggcaat gcccacggca aggatgtgga cgccttcatc agactgaccg
     3541 tggacgacat gctgaccgac accgagcgga gaatcaagag attcaaggac gaccggaagt
     3601 ccattcggag cgccgacaac aagatgggaa agagaggctt caagcagatc tccacaggca
     3661 agctggccga cttcctggcc aaggacatcg tgctgtttca gcccagcgtg aacgatggcg
     3721 agaacaagat caccggcctg aactaccgga tcatgcagag cgccattgcc gtgtacgata
     3781 gcggcgacga ttacgaggcc aagcagcagt tcaagctgat gttcgagaag gcccggctga
     3841 tcggcaaggg cacaacagag cctcatccat ttctgtacaa ggtgttcgcc cgcagcatcc
     3901 ccgccaatgc cgtcgagttc tacgagcgct acctgatcga gcggaagttc tacctgaccg
     3961 gcctgtccaa cgagatcaag aaaggcaaca gagtggatgt gcccttcatc cggcgggacc
     4021 agaacaagtg gaaaacaccc gccatgaaga ccctgggcag aatctacagc gaggatctgc
     4081 ccgtggaact gcccagacag atgttcgaca atgagatcaa gtcccacctg aagtccctgc
     4141 cacagatgga aggcatcgac ttcaacaatg ccaacgtgac ctatctgatc gccgagtaca
     4201 tgaagagagt gctggacgac gacttccaga ccttctacca gtggaaccgc aactaccggt
     4261 acatggacat gcttaagggc gagtacgaca gaaagggctc cctgcagcac tgcttcacca
     4321 gcgtggaaga gagagaaggc ctctggaaag agcgggcctc cagaacagag cggtacagaa
     4381 agcaggccag caacaagatc cgcagcaacc ggcagatgag aaacgccagc agcgaagaga
     4441 tcgagacaat cctggataag cggctgagca acagccggaa cgagtaccag aaaagcgaga
     4501 aagtgatccg gcgctacaga gtgcaggatg ccctgctgtt tctgctggcc aaaaagaccc
     4561 tgaccgaact ggccgatttc gacggcgaga ggttcaaact gaaagaaatc atgcccgacg
     4621 ccgagaaggg aatcctgagc gagatcatgc ccatgagctt caccttcgag aaaggcggca
     4681 agaagtacac catcaccagc gagggcatga agctgaagaa ctacggcgac ttctttgtgc
     4741 tggctagcga caagaggatc ggcaacctgc tggaactcgt gggcagcgac atcgtgtcca
     4801 aagaggatat catggaagag ttcaacaaat acgaccagtg caggcccgag atcagctcca
     4861 tcgtgttcaa cctggaaaag tgggccttcg acacataccc cgagctgtct gccagagtgg
     4921 accgggaaga gaaggtggac ttcaagagca tcctgaaaat cctgctgaac aacaagaaca
     4981 tcaacaaaga gcagagcgac atcctgcgga agatccggaa cgccttcgat cacaacaatt
     5041 accccgacaa aggcgtggtg gaaatcaagg ccctgcctga gatcgccatg agcatcaaga
     5101 aggcctttgg ggagtacgcc atcatgaagg gatccggact gcctccactt gaaagactga
     5161 cactggcggc cgctatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     5221 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     5281 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     5341 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     5401 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     5461 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     5521 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     5581 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     5641 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     5701 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     5761 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     5821 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     5881 agtaagcggc cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     5941 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     6001 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     6061 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     6121 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     6181 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     6241 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     6301 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     6361 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
     6421 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6481 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6541 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6601 gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct
     6661 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     6721 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     6781 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca
     6841 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     6901 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     6961 cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     7021 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     7081 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     7141 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     7201 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     7261 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     7321 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     7381 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     7441 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     7501 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     7561 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     7621 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     7681 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     7741 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     7801 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7861 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     7921 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     7981 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     8041 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     8101 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     8161 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     8221 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     8281 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     8341 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     8401 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     8461 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     8521 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     8581 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     8641 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     8701 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     8761 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     8821 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     8881 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     8941 tgccacctgg
//
