LOCUS       sequence-383155-        7451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express hfCas13d.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7451)
  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 7451)
  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-U6-DR30(sapI)-CMV-intron-hfCas13d-pA
FEATURES             Location/Qualifiers
     source          1..7451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      32..620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     521..540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   682..811
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        826..1074
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     826..846
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     997..1016
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1076..1105
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        1205..1508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1509..1712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1663..1683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1709..1733
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1861..2239
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             2322..2342
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2352..5249
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILVEYITNVVYVVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             5259..5279
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5289..5315
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5330..5378
                     /note="synthetic polyadenylation signal"
     repeat_region   5395..5535
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5395..5524
                     /label=AAV2 ITR
     rep_origin      5610..6065
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5697..5716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5907..5928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6082..6101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6201..6223
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6261..6279)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6347..6451
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6452..6520,6521..7312)
                     /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(6670..6689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
       61 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      121 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
      181 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      241 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      301 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      361 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      421 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      481 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      541 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      601 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      661 gctggccttt tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg
      721 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
      781 ggagtggcca actccatcac taggggttcc tgcggccgca ctagcgaggg cctatttccc
      841 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
      901 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      961 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1021 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgaaccc
     1081 ctaccaactg gtcggggttt gaaacggaag agcgagctct tctttttttt ctgtacaacg
     1141 cgtctagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     1201 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1261 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     1321 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     1381 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     1441 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     1501 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     1561 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     1621 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     1681 gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
     1741 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg
     1801 gattcgaatc ccggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac
     1861 gtaagtaccg cctatagagt ctataggccc acaaaaaatg ctttcttctt ttaatatact
     1921 tttttgttta tcttatttct aatactttcc ctaatctctt tctttcaggg caataatgat
     1981 acaatgtatc atgcctcttt gcaccattct aaagaataac agtgataatt tctgggttaa
     2041 ggcaatagca atatttctgc atataaatat ttctgcatat aaattgtaac tgatgtaaga
     2101 ggtttcatat tgctaatagc agctacaatc cagctaccat tctgctttta ttttatggtt
     2161 gggataaggc tggattattc tgagtccaag ctaggccctt ttgctaatca tgttcatacc
     2221 tcttatcttc ctcccacagc tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt
     2281 tggcaaagaa ttgggattcg aacatcgatg ccaccatgag ccccaagaag aagagaaagg
     2341 tggaggccag catcgaaaaa aaaaagtcct tcgccaaggg catgggcgtg aagtccacac
     2401 tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga aggcagcgac gccaggctgg
     2461 aaaagatcgt ggagggcgac agcatcagga gcgtgaatga gggcgaggcc ttcagcgctg
     2521 aaatggccga taaaaacgcc ggctataaga tcggcaacgc caaattcagc catcctaagg
     2581 gctacgccgt ggtggctaac aaccctctgt atacaggacc cgtccagcag gatatgctcg
     2641 gcctgaagga aactctggaa aagaggtact tcggcgagag cgctgatggc aatgacaata
     2701 tttgtatcca ggtgatccat aacatcctgg acattgaaaa aatcctcgtg gaatacatta
     2761 ccaacgtggt gtacgtggtc aacaatatct ccggcctgga taaggacatt attggattcg
     2821 gcaagttctc cacagtgtat acctacgacg aattcaaaga ccccgagcac catagggccg
     2881 ctttcaacaa taacgataag ctcatcaacg ccatcaaggc ccagtatgac gagttcgaca
     2941 acttcctcga taaccccaga ctcggctatt tcggccaggc ctttttcagc aaggagggca
     3001 gaaattacat catcaattac ggcaacgaat gctatgacat tctggccctc ctgagcggac
     3061 tgaggcactg ggtggtccat aacaacgaag aagagtccag gatctccagg acctggctct
     3121 acaacctcga taagaacctc gacaacgaat acatctccac cctcaactac ctctacgaca
     3181 ggatcaccaa tgagctgacc aactccttct ccaagaactc cgccgccaac gtgaactata
     3241 ttgccgaaac tctgggaatc aaccctgccg aattcgccga acaatatttc agattcagca
     3301 ttatgaaaga gcagaaaaac ctcggattca atatcaccaa gctcagggaa gtgatgctgg
     3361 acaggaagga tatgtccgag atcaggaaaa atcataaggt gttcgactcc atcaggacca
     3421 aggtctacac catgatggac tttgtgattt ataggtatta catcgaagag gatgccaagg
     3481 tggctgccgc caataagtcc ctccccgata atgagaagtc cctgagcgag aaggatatct
     3541 ttgtgattaa cctgaggggc tccttcaacg acgaccagaa ggatgccctc tactacgatg
     3601 aagctaatag aatttggaga aagctcgaaa atatcatgca caacatcaag gaatttaggg
     3661 gaaacaagac aagagagtat aagaagaagg acgcccctag actgcccaga atcctgcccg
     3721 ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc cctgaccatg ttcctggatg
     3781 gcaaggagat caacgacctc ctgaccaccc tgattaataa attcgataac atccagagct
     3841 tcctgaaggt gatgcctctc atcggagtca acgctaagtt cgtggaggaa tacgcctttt
     3901 tcaaagactc cgccaagatc gccgatgagc tgaggctgat caagtccttc gctagaatgg
     3961 gagaacctat tgccgatgcc aggagggcca tgtatatcga cgccatccgt attttaggaa
     4021 ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac cttttccctg gacgagaacg
     4081 gaaacaagct caagaaaggc aagcacggca tgagaaattt cattattaat aacgtgatca
     4141 gcaataaaag gttccactac ctgatcagat acggtgatcc tgcccacctc catgagatcg
     4201 ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat cgctgacatc cagaaaaaac
     4261 agggccagaa cggcaagaac cagatcgaca ggtactacga aacttgtatc ggaaaggata
     4321 agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa gatcatcacc ggaatgaact
     4381 acgaccaatt cgacaagaaa aggagcgtca ttgaggacac cggcagggaa aacgccgaga
     4441 gggagaagtt taaaaagatc atcagcctgt acctcaccgt gatctaccac atcctcaaga
     4501 atattgtcaa tatcaacgcc aggtacgtca tcggattcca ttgcgtcgag cgtgatgctc
     4561 aactgtacaa ggagaaaggc tacgacatca atctcaagaa actggaagag aagggattca
     4621 gctccgtcac caagctctgc gctggcattg atgaaactgc ccccgataag agaaaggacg
     4681 tggaaaagga gatggctgaa agagccaagg agagcattga cagcctcgag agcgccaacc
     4741 ccaagctgta tgccaattac atcaaataca gcgacgagaa gaaagccgag gagttcacca
     4801 ggcagattaa cagggagaag gccaaaaccg ccctgaacgc ctacctgagg aacaccaagt
     4861 ggaatgtgat catcagggag gacctcctga gaattgacaa caagacatgt accctgttca
     4921 gaaacaaggc cgtccacctg gaagtggcca ggtatgtcca cgcctatatc aacgacattg
     4981 ccgaggtcaa ttcctacttc caactgtacc attacatcat gcagagaatt atcatgaatg
     5041 agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt cgacgctgtg aatgacgaga
     5101 agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc tttcggctac tgtatcccca
     5161 ggtttaagaa cctgagcatc gaggccctgt tcgataggaa cgaggccgcc aagttcgaca
     5221 aggagaaaaa gaaggtgtcc ggcaattccg gatccggacc taagaaaaag aggaaggtgg
     5281 cggccgctta cccatacgat gttccagatt acgcttaggc taggaattca ataaaagatc
     5341 tttattttca ttagatctgt gtgttggttt tttgtgtgcg gaccgagcgg ccgcaggaac
     5401 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     5461 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5521 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5581 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     5641 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     5701 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5761 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5821 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5881 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5941 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     6001 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     6061 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     6121 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6181 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6241 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     6301 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6361 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6421 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6481 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6541 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6601 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6661 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6721 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6781 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6841 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6901 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6961 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7021 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7081 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7141 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7201 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7261 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7321 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7381 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     7441 ccactgagcg t
//
