LOCUS       sequence-383151-        7133 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 7133)
  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 7133)
  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-CMV-intron-hfCas13d-pA
FEATURES             Location/Qualifiers
     source          1..7133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   60..189
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        265..568
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        569..772
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     723..743
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     769..793
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          921..1299
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1382..1402
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1412..4309
                     /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             4319..4339
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4349..4375
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4390..4438
                     /note="synthetic polyadenylation signal"
     repeat_region   4455..4595
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4455..4584
                     /label=AAV2 ITR
     rep_origin      4670..5125
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4757..4776)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4967..4988
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5142..5161)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5261..5283
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5321..5339)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5407..5511
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5512..5580,5581..6372)
                     /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(5730..5749)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6543..7131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7032..7051
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
       61 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
      121 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
      181 ggggttcctg cggccgcacg cgtctagtta ttaatagtaa tcaattacgg ggtcattagt
      241 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      301 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      361 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      421 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      481 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      541 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      601 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      661 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      721 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
      781 gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg
      841 ggaccgatcc agcctccgcg gattcgaatc ccggccggga acggtgcatt ggaacgcgga
      901 ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc acaaaaaatg
      961 ctttcttctt ttaatatact tttttgttta tcttatttct aatactttcc ctaatctctt
     1021 tctttcaggg caataatgat acaatgtatc atgcctcttt gcaccattct aaagaataac
     1081 agtgataatt tctgggttaa ggcaatagca atatttctgc atataaatat ttctgcatat
     1141 aaattgtaac tgatgtaaga ggtttcatat tgctaatagc agctacaatc cagctaccat
     1201 tctgctttta ttttatggtt gggataaggc tggattattc tgagtccaag ctaggccctt
     1261 ttgctaatca tgttcatacc tcttatcttc ctcccacagc tcctgggcaa cgtgctggtc
     1321 tgtgtgctgg cccatcactt tggcaaagaa ttgggattcg aacatcgatg ccaccatgag
     1381 ccccaagaag aagagaaagg tggaggccag catcgaaaaa aaaaagtcct tcgccaaggg
     1441 catgggcgtg aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga
     1501 aggcagcgac gccaggctgg aaaagatcgt ggagggcgac agcatcagga gcgtgaatga
     1561 gggcgaggcc ttcagcgctg aaatggccga taaaaacgcc ggctataaga tcggcaacgc
     1621 caaattcagc catcctaagg gctacgccgt ggtggctaac aaccctctgt atacaggacc
     1681 cgtccagcag gatatgctcg gcctgaagga aactctggaa aagaggtact tcggcgagag
     1741 cgctgatggc aatgacaata tttgtatcca ggtgatccat aacatcctgg acattgaaaa
     1801 aatcctcgtg gaatacatta ccaacgtggt gtacgtggtc aacaatatct ccggcctgga
     1861 taaggacatt attggattcg gcaagttctc cacagtgtat acctacgacg aattcaaaga
     1921 ccccgagcac catagggccg ctttcaacaa taacgataag ctcatcaacg ccatcaaggc
     1981 ccagtatgac gagttcgaca acttcctcga taaccccaga ctcggctatt tcggccaggc
     2041 ctttttcagc aaggagggca gaaattacat catcaattac ggcaacgaat gctatgacat
     2101 tctggccctc ctgagcggac tgaggcactg ggtggtccat aacaacgaag aagagtccag
     2161 gatctccagg acctggctct acaacctcga taagaacctc gacaacgaat acatctccac
     2221 cctcaactac ctctacgaca ggatcaccaa tgagctgacc aactccttct ccaagaactc
     2281 cgccgccaac gtgaactata ttgccgaaac tctgggaatc aaccctgccg aattcgccga
     2341 acaatatttc agattcagca ttatgaaaga gcagaaaaac ctcggattca atatcaccaa
     2401 gctcagggaa gtgatgctgg acaggaagga tatgtccgag atcaggaaaa atcataaggt
     2461 gttcgactcc atcaggacca aggtctacac catgatggac tttgtgattt ataggtatta
     2521 catcgaagag gatgccaagg tggctgccgc caataagtcc ctccccgata atgagaagtc
     2581 cctgagcgag aaggatatct ttgtgattaa cctgaggggc tccttcaacg acgaccagaa
     2641 ggatgccctc tactacgatg aagctaatag aatttggaga aagctcgaaa atatcatgca
     2701 caacatcaag gaatttaggg gaaacaagac aagagagtat aagaagaagg acgcccctag
     2761 actgcccaga atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc
     2821 cctgaccatg ttcctggatg gcaaggagat caacgacctc ctgaccaccc tgattaataa
     2881 attcgataac atccagagct tcctgaaggt gatgcctctc atcggagtca acgctaagtt
     2941 cgtggaggaa tacgcctttt tcaaagactc cgccaagatc gccgatgagc tgaggctgat
     3001 caagtccttc gctagaatgg gagaacctat tgccgatgcc aggagggcca tgtatatcga
     3061 cgccatccgt attttaggaa ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac
     3121 cttttccctg gacgagaacg gaaacaagct caagaaaggc aagcacggca tgagaaattt
     3181 cattattaat aacgtgatca gcaataaaag gttccactac ctgatcagat acggtgatcc
     3241 tgcccacctc catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat
     3301 cgctgacatc cagaaaaaac agggccagaa cggcaagaac cagatcgaca ggtactacga
     3361 aacttgtatc ggaaaggata agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa
     3421 gatcatcacc ggaatgaact acgaccaatt cgacaagaaa aggagcgtca ttgaggacac
     3481 cggcagggaa aacgccgaga gggagaagtt taaaaagatc atcagcctgt acctcaccgt
     3541 gatctaccac atcctcaaga atattgtcaa tatcaacgcc aggtacgtca tcggattcca
     3601 ttgcgtcgag cgtgatgctc aactgtacaa ggagaaaggc tacgacatca atctcaagaa
     3661 actggaagag aagggattca gctccgtcac caagctctgc gctggcattg atgaaactgc
     3721 ccccgataag agaaaggacg tggaaaagga gatggctgaa agagccaagg agagcattga
     3781 cagcctcgag agcgccaacc ccaagctgta tgccaattac atcaaataca gcgacgagaa
     3841 gaaagccgag gagttcacca ggcagattaa cagggagaag gccaaaaccg ccctgaacgc
     3901 ctacctgagg aacaccaagt ggaatgtgat catcagggag gacctcctga gaattgacaa
     3961 caagacatgt accctgttca gaaacaaggc cgtccacctg gaagtggcca ggtatgtcca
     4021 cgcctatatc aacgacattg ccgaggtcaa ttcctacttc caactgtacc attacatcat
     4081 gcagagaatt atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt
     4141 cgacgctgtg aatgacgaga agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc
     4201 tttcggctac tgtatcccca ggtttaagaa cctgagcatc gaggccctgt tcgataggaa
     4261 cgaggccgcc aagttcgaca aggagaaaaa gaaggtgtcc ggcaattccg gatccggacc
     4321 taagaaaaag aggaaggtgg cggccgctta cccatacgat gttccagatt acgcttaggc
     4381 taggaattca ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgcg
     4441 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4501 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4561 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4621 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4681 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4741 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4801 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4861 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4921 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4981 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     5041 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5101 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     5161 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5221 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5281 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5341 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5401 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5461 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5521 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5581 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5641 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5701 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5761 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5821 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5881 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5941 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6001 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6061 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6121 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6181 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6241 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6301 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6361 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6421 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6481 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6541 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6601 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6661 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6721 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6781 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6841 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6901 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6961 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7021 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7081 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaa
//
