LOCUS       sequence-383150-        7402 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 7402)
  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 7402)
  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-DIO-hfCas13d-pA
FEATURES             Location/Qualifiers
     source          1..7402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   21..161
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   21..150
                     /label=AAV2 ITR
     rep_origin      236..691
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(323..342)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     533..554
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(708..727)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     827..849
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(887..905)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        973..1077
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1078..1146,1147..1938)
                     /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(1296..1315)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2109..2697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2598..2617
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2759..2888
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2968..3271
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3272..3475
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3426..3446
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3472..3496
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          3624..4002
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     protein_bind    complement(4054..4087)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(4138..4171)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4183..4209)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4219..4239)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4249..7146)
                     /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             complement(7156..7176)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    7195..7228
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    7279..7312
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(7365..7384)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 tcactggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
       61 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
      121 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
      181 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
      241 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      301 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      361 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      421 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
      481 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      541 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
      601 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      661 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
      721 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
      781 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
      841 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
      901 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
      961 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1021 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1081 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1141 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1201 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1261 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1321 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1381 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1441 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1501 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1561 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1621 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1681 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1741 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1801 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1861 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1921 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1981 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2041 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2101 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2161 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2221 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2281 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2341 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2401 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2461 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2521 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2581 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2641 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2701 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     2761 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
     2821 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2881 gggttcctgc ggccgcacgc gtggagctag ttattaatag taatcaatta cggggtcatt
     2941 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     3001 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     3061 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     3121 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     3181 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     3241 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     3301 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     3361 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     3421 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt
     3481 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     3541 ccgggaccga tccagcctcc gcggattcga atcccggccg ggaacggtgc attggaacgc
     3601 ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg cccacaaaaa
     3661 atgctttctt cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct
     3721 ctttctttca gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat
     3781 aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca
     3841 tataaattgt aactgatgta agaggtttca tattgctaat agcagctaca atccagctac
     3901 cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc
     3961 cttttgctaa tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg
     4021 gtctgtgtgc tggcccatca cgtcgactcc ggaataactt cgtataggat actttatacg
     4081 aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa cctcttaata
     4141 acttcgtata gcatacatta tacgaagtta tggcgcgccc taagcgtaat ctggaacatc
     4201 gtatgggtac gcagcggcca ccttcctctt tttcttaggt ccggatccgg aattgccgga
     4261 caccttcttt ttctccttgt cgaacttggc ggcctcgttc ctatcgaaca gggcctcgat
     4321 gctcaggttc ttaaacctgg ggatacagta gccgaaaggc acacacagca gtttcaggag
     4381 cctatcgttg tacttcttct cgtcattcac agcgtcgaag tactcggaca cctttccgct
     4441 gcttttctcg tacctctcat tcatgataat tctctgcatg atgtaatggt acagttggaa
     4501 gtaggaattg acctcggcaa tgtcgttgat ataggcgtgg acatacctgg ccacttccag
     4561 gtggacggcc ttgtttctga acagggtaca tgtcttgttg tcaattctca ggaggtcctc
     4621 cctgatgatc acattccact tggtgttcct caggtaggcg ttcagggcgg ttttggcctt
     4681 ctccctgtta atctgcctgg tgaactcctc ggctttcttc tcgtcgctgt atttgatgta
     4741 attggcatac agcttggggt tggcgctctc gaggctgtca atgctctcct tggctctttc
     4801 agccatctcc ttttccacgt cctttctctt atcgggggca gtttcatcaa tgccagcgca
     4861 gagcttggtg acggagctga atcccttctc ttccagtttc ttgagattga tgtcgtagcc
     4921 tttctccttg tacagttgag catcacgctc gacgcaatgg aatccgatga cgtacctggc
     4981 gttgatattg acaatattct tgaggatgtg gtagatcacg gtgaggtaca ggctgatgat
     5041 ctttttaaac ttctccctct cggcgttttc cctgccggtg tcctcaatga cgctcctttt
     5101 cttgtcgaat tggtcgtagt tcattccggt gatgatcttt gtgagagcgt ccaccttttc
     5161 gctcacgctc ttgcccttat cctttccgat acaagtttcg tagtacctgt cgatctggtt
     5221 cttgccgttc tggccctgtt ttttctggat gtcagcgatc ctgccgagca cgaacttcac
     5281 cacggcctcg tttttggcga tctcatggag gtgggcagga tcaccgtatc tgatcaggta
     5341 gtggaacctt ttattgctga tcacgttatt aataatgaaa tttctcatgc cgtgcttgcc
     5401 tttcttgagc ttgtttccgt tctcgtccag ggaaaaggtg tcggcgaggg ccttgagctc
     5461 atcataggac aggttggttc ctaaaatacg gatggcgtcg atatacatgg ccctcctggc
     5521 atcggcaata ggttctccca ttctagcgaa ggacttgatc agcctcagct catcggcgat
     5581 cttggcggag tctttgaaaa aggcgtattc ctccacgaac ttagcgttga ctccgatgag
     5641 aggcatcacc ttcaggaagc tctggatgtt atcgaattta ttaatcaggg tggtcaggag
     5701 gtcgttgatc tccttgccat ccaggaacat ggtcagggca tacatgagtt tgctgaaggc
     5761 ggaaacatca cggccagcgg gcaggattct gggcagtcta ggggcgtcct tcttcttata
     5821 ctctcttgtc ttgtttcccc taaattcctt gatgttgtgc atgatatttt cgagctttct
     5881 ccaaattcta ttagcttcat cgtagtagag ggcatccttc tggtcgtcgt tgaaggagcc
     5941 cctcaggtta atcacaaaga tatccttctc gctcagggac ttctcattat cggggaggga
     6001 cttattggcg gcagccacct tggcatcctc ttcgatgtaa tacctataaa tcacaaagtc
     6061 catcatggtg tagaccttgg tcctgatgga gtcgaacacc ttatgatttt tcctgatctc
     6121 ggacatatcc ttcctgtcca gcatcacttc cctgagcttg gtgatattga atccgaggtt
     6181 tttctgctct ttcataatgc tgaatctgaa atattgttcg gcgaattcgg cagggttgat
     6241 tcccagagtt tcggcaatat agttcacgtt ggcggcggag ttcttggaga aggagttggt
     6301 cagctcattg gtgatcctgt cgtagaggta gttgagggtg gagatgtatt cgttgtcgag
     6361 gttcttatcg aggttgtaga gccaggtcct ggagatcctg gactcttctt cgttgttatg
     6421 gaccacccag tgcctcagtc cgctcaggag ggccagaatg tcatagcatt cgttgccgta
     6481 attgatgatg taatttctgc cctccttgct gaaaaaggcc tggccgaaat agccgagtct
     6541 ggggttatcg aggaagttgt cgaactcgtc atactgggcc ttgatggcgt tgatgagctt
     6601 atcgttattg ttgaaagcgg ccctatggtg ctcggggtct ttgaattcgt cgtaggtata
     6661 cactgtggag aacttgccga atccaataat gtccttatcc aggccggaga tattgttgac
     6721 cacgtacacc acgttggtaa tgtattccac gaggattttt tcaatgtcca ggatgttatg
     6781 gatcacctgg atacaaatat tgtcattgcc atcagcgctc tcgccgaagt acctcttttc
     6841 cagagtttcc ttcaggccga gcatatcctg ctggacgggt cctgtataca gagggttgtt
     6901 agccaccacg gcgtagccct taggatggct gaatttggcg ttgccgatct tatagccggc
     6961 gtttttatcg gccatttcag cgctgaaggc ctcgccctca ttcacgctcc tgatgctgtc
     7021 gccctccacg atcttttcca gcctggcgtc gctgccttcg gcgaaggttg tcatgtacac
     7081 tttggagccg gacacgagtg tggacttcac gcccatgccc ttggcgaagg actttttttt
     7141 ttcgatgctg gcctccacct ttctcttctt cttggggctc atggtggcgc tagcataact
     7201 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc
     7261 tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga
     7321 tactagtctc gagtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     7381 tttcacaaat aaagcatttt tt
//
