LOCUS       sequence-380388-        6737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express DjCas13d.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6737)
  AUTHORS   Back F, Sandoval A, Vu LM, Hong VM, Bhaskara A, Rodriguez SR, 
            O'Brien JT, Kolber BJ, Kroener S, Ploski JE
  TITLE     Adeno-associated viral vector resource for the RNA-targeting Cas13d:
            A comparison of high-fidelity variants, DjCas13d and hfCas13d.
  JOURNAL   Mol Ther Methods Clin Dev. 2025 Aug 20;33(4):101565. doi: 
            10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11.
  PUBMED    41020266
REFERENCE   2  (bases 1 to 6737)
  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: "Mol Ther 
            Methods Clin Dev."; date: "2025-08-20"; volume: "33(4):101565. doi";
            pages: " 10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11"
COMMENT     Sequence Label: pAAV-EFS-DjCas13d-T2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..6737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     513..532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   674..803
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        857..1068
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1107..1127
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3777..3797
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3807..3833
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3849..3902
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3903..4610
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4045..4063)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4313..4332
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4617..4665
                     /note="synthetic polyadenylation signal"
     repeat_region   4673..4813
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4673..4802
                     /label=AAV2 ITR
     rep_origin      4888..5343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4975..4994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5185..5206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5360..5379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5479..5501
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5539..5557)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5625..5729
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5730..5798,5799..6590)
                     /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(5948..5967)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg
      721 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
      781 caactccatc actaggggtt cctgcggcct ctagagctag gcgtcctaga gtcgagtggc
      841 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
      901 ggggtcggca attgaacggg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
      961 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     1021 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc
     1081 ggaccggtgc tagcgccacc atgagcccca agaagaagag aaaggtggag gccagcatca
     1141 agaagcaaaa atccaagaaa acggtttcta aaacctccgg attgaaagag gcactttctg
     1201 tgcaggggac tgtcataatg acgtcttttg ggaaggggaa catggcgaat ctctcttaca
     1261 agatcccgag ctctcaaaag ccccaaaatc tcaatagtag cgccggcttg aagaacgtcg
     1321 aagtatctgg taaaaagatt aagttccaag gtcgacaccc gaagatcgcc actaccgaca
     1381 accccctgtt taagccgcag ccaggtatgg accttctgtg cctgaaagac aaactcgaaa
     1441 tgcactattt tgggaagact ttcgacgaca atatccacat acaattgata tatcaaatac
     1501 tggatattga aaagatcctt gcggttcatg ttaacaacat cgtattcaca ctggacaatg
     1561 tcctgcatcc gcaaaaggaa gagctgactg aagatttcat tggggcaggg ggctggcgga
     1621 ttaatctcga ttaccaaact cttagagggc agactaacaa atatgatagg tttaaaaact
     1681 acattaaaag aaaagaactt ctttatttcg gggaggcatt ttaccatgaa aacgagagac
     1741 ggtatgagga agacatcttc gcaatactta ccctgctgtc tgcccttaga cagttttgct
     1801 ttcattccga tctgagtagt gatgagagcg accacgtgaa ttcattctgg ctgtatcaac
     1861 tcgaggacca actgtctgac gagttcaagg agactctcag tattttgtgg gaagaggtca
     1921 ccgaaaggat agatagtgaa ttccttaaaa ctaatacagt aaacctccat attttgtgtc
     1981 atgttttccc gaaagagagt aaggagacga ttgtacgcgc ttattatgaa ttcctgatca
     2041 agaagtcctt taaaaatatg ggatttagca tcaagaaatt gcgagagatt atgctcgagc
     2101 aatcagatct taagagtttc aaagaggata agtacaactc cgtcagggct aaactgtata
     2161 agctcttcga ctttattata acctattact acgaccacca tgcttttgag aaggaggcgc
     2221 tcgtgagctc cttgcgaagt tcactcactg aggaaaacaa ggaagagatt tacatcaaaa
     2281 cggcccggac actcgcgagc gctcttgggg ccgatttcaa gaaagccgct gccgatgtaa
     2341 atgccaaaaa cataagggac tatcagaaga aggcgaacga ctataggata agttttgaag
     2401 atattaaaat agggaacaca ggcattgggt atttctcaga gctcatatac atgctcaccc
     2461 ttctcctcga tggaaaggag ataaacgacc tcctcacaac cctgatcaac aaatttgata
     2521 atataatctc cttcatagac atcctgaaga aactcaattt ggaatttaaa ttcaagcctg
     2581 aatacgcgga cttctttaac atgaccaact gcaggtatac tctcgaagag ctgcgggtta
     2641 tcaacagtat cgctagaatg caaaagccaa gcgccgacgc ccgaaagatc atgtatcgcg
     2701 atgctctgcg catattggga atggacaatc ggccggatga agagattgat cgggaactcg
     2761 aaaggacaat gcccgtgggt gcggatggca aatttatcaa aggtaaacag ggctttcgca
     2821 atttcattgc ttccaacgtt attgaatctt cacgcttcca ttacctcgta cggtataata
     2881 atccacacaa gacacggact cttgtgaaaa accctaatgt agtaaaattt gtgctcgaag
     2941 ggattccgga gacacaaata aaacgatact ttgatgtttg taaaggtcaa gagatcccac
     3001 cgacgtctga caagtccgca cagatagacg ttcttgcccg aataattagt tcagtagact
     3061 ataaaatatt tgaagacgta ccccaaagtg caaagatcaa taaagatgat cctagccgaa
     3121 acttcagtga cgcgctgaaa aaacagcggt accaggcaat tgtaagtctt tatttgactg
     3181 ttatgtacct tataaccaaa aacctggtgt atgtcaatag taggtatgtg attgcatttc
     3241 attgcctgga acgagacgca ttccttcacg gtgtgacgct ccctaagatg aataaaaaaa
     3301 ttgtgtacag tcaattgact actcacctct tgactgacaa aaattacact acgtacggcc
     3361 acctgaagaa ccagaaagga caccgcaaat ggtacgttct tgtcaaaaat aaccttcaga
     3421 atagcgacat aacagctgtg agttcattcc gcaacatagt tgctcacata tccgttgtga
     3481 ggaatagtaa tgaatacata agcgggatcg gagaactgca ctcttacttc gaactttatc
     3541 actatctcgt tcaatcaatg atcgcaaaga ataattggta tgataccagc caccagccga
     3601 agaccgccga atatctgaac aatctgaaaa agcaccacac ttattgtaag gactttgtaa
     3661 aagcgtactg tatcccgttc ggatacgtcg tgccgcggta caaaaacctc acgataaatg
     3721 aactttttga tcgaaataac ccgaatcccg aaccaaaaga ggaagtggga tccggaccta
     3781 agaaaaagag gaaggtggcc gctgcgtacc catacgatgt tccagattac gctgccagcg
     3841 gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     3901 cagtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     3961 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     4021 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     4081 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     4141 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     4201 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     4261 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     4321 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     4381 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     4441 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     4501 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     4561 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa aagcttaata
     4621 aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgcggcc gcaggaaccc
     4681 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4741 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4801 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4861 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4921 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4981 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5041 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5101 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5161 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     5221 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     5281 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5341 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5401 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5461 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5521 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5581 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5641 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5701 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5761 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5821 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5881 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5941 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6001 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6061 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6121 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6181 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6241 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6301 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6361 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6421 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     6481 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6541 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6601 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6661 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6721 actgagcgtc agacccc
//
