LOCUS       sequence-463798-        7152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express hfCas13d from a EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7152)
  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 7152)
  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-EF1a-hfCas13d-pa
FEATURES             Location/Qualifiers
     source          1..7152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        795..1973
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1026..1964
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1921..1941
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1992..2012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2022..4919
                     /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             4929..4949
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4959..4985
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5000..5048
                     /note="synthetic polyadenylation signal"
     repeat_region   5065..5205
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5065..5194
                     /label=AAV2 ITR
     rep_origin      5280..5735
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5367..5386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5577..5598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5752..5771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5871..5893
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5931..5949)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6017..6121
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6122..6190,6191..6982)
                     /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(6340..6359)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
      841 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
      901 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
      961 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     1021 cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
     1081 gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg
     1141 ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt
     1201 gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg
     1261 cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
     1321 cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
     1381 cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga
     1441 ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc
     1501 ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg
     1561 cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
     1621 gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
     1681 aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt
     1741 ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg
     1801 ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt
     1861 ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
     1921 tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaatcgatg
     1981 ccaccatgag ccccaagaag aagagaaagg tggaggccag catcgaaaaa aaaaagtcct
     2041 tcgccaaggg catgggcgtg aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa
     2101 ccttcgccga aggcagcgac gccaggctgg aaaagatcgt ggagggcgac agcatcagga
     2161 gcgtgaatga gggcgaggcc ttcagcgctg aaatggccga taaaaacgcc ggctataaga
     2221 tcggcaacgc caaattcagc catcctaagg gctacgccgt ggtggctaac aaccctctgt
     2281 atacaggacc cgtccagcag gatatgctcg gcctgaagga aactctggaa aagaggtact
     2341 tcggcgagag cgctgatggc aatgacaata tttgtatcca ggtgatccat aacatcctgg
     2401 acattgaaaa aatcctcgtg gaatacatta ccaacgtggt gtacgtggtc aacaatatct
     2461 ccggcctgga taaggacatt attggattcg gcaagttctc cacagtgtat acctacgacg
     2521 aattcaaaga ccccgagcac catagggccg ctttcaacaa taacgataag ctcatcaacg
     2581 ccatcaaggc ccagtatgac gagttcgaca acttcctcga taaccccaga ctcggctatt
     2641 tcggccaggc ctttttcagc aaggagggca gaaattacat catcaattac ggcaacgaat
     2701 gctatgacat tctggccctc ctgagcggac tgaggcactg ggtggtccat aacaacgaag
     2761 aagagtccag gatctccagg acctggctct acaacctcga taagaacctc gacaacgaat
     2821 acatctccac cctcaactac ctctacgaca ggatcaccaa tgagctgacc aactccttct
     2881 ccaagaactc cgccgccaac gtgaactata ttgccgaaac tctgggaatc aaccctgccg
     2941 aattcgccga acaatatttc agattcagca ttatgaaaga gcagaaaaac ctcggattca
     3001 atatcaccaa gctcagggaa gtgatgctgg acaggaagga tatgtccgag atcaggaaaa
     3061 atcataaggt gttcgactcc atcaggacca aggtctacac catgatggac tttgtgattt
     3121 ataggtatta catcgaagag gatgccaagg tggctgccgc caataagtcc ctccccgata
     3181 atgagaagtc cctgagcgag aaggatatct ttgtgattaa cctgaggggc tccttcaacg
     3241 acgaccagaa ggatgccctc tactacgatg aagctaatag aatttggaga aagctcgaaa
     3301 atatcatgca caacatcaag gaatttaggg gaaacaagac aagagagtat aagaagaagg
     3361 acgcccctag actgcccaga atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac
     3421 tcatgtatgc cctgaccatg ttcctggatg gcaaggagat caacgacctc ctgaccaccc
     3481 tgattaataa attcgataac atccagagct tcctgaaggt gatgcctctc atcggagtca
     3541 acgctaagtt cgtggaggaa tacgcctttt tcaaagactc cgccaagatc gccgatgagc
     3601 tgaggctgat caagtccttc gctagaatgg gagaacctat tgccgatgcc aggagggcca
     3661 tgtatatcga cgccatccgt attttaggaa ccaacctgtc ctatgatgag ctcaaggccc
     3721 tcgccgacac cttttccctg gacgagaacg gaaacaagct caagaaaggc aagcacggca
     3781 tgagaaattt cattattaat aacgtgatca gcaataaaag gttccactac ctgatcagat
     3841 acggtgatcc tgcccacctc catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc
     3901 tcggcaggat cgctgacatc cagaaaaaac agggccagaa cggcaagaac cagatcgaca
     3961 ggtactacga aacttgtatc ggaaaggata agggcaagag cgtgagcgaa aaggtggacg
     4021 ctctcacaaa gatcatcacc ggaatgaact acgaccaatt cgacaagaaa aggagcgtca
     4081 ttgaggacac cggcagggaa aacgccgaga gggagaagtt taaaaagatc atcagcctgt
     4141 acctcaccgt gatctaccac atcctcaaga atattgtcaa tatcaacgcc aggtacgtca
     4201 tcggattcca ttgcgtcgag cgtgatgctc aactgtacaa ggagaaaggc tacgacatca
     4261 atctcaagaa actggaagag aagggattca gctccgtcac caagctctgc gctggcattg
     4321 atgaaactgc ccccgataag agaaaggacg tggaaaagga gatggctgaa agagccaagg
     4381 agagcattga cagcctcgag agcgccaacc ccaagctgta tgccaattac atcaaataca
     4441 gcgacgagaa gaaagccgag gagttcacca ggcagattaa cagggagaag gccaaaaccg
     4501 ccctgaacgc ctacctgagg aacaccaagt ggaatgtgat catcagggag gacctcctga
     4561 gaattgacaa caagacatgt accctgttca gaaacaaggc cgtccacctg gaagtggcca
     4621 ggtatgtcca cgcctatatc aacgacattg ccgaggtcaa ttcctacttc caactgtacc
     4681 attacatcat gcagagaatt atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt
     4741 ccgagtactt cgacgctgtg aatgacgaga agaagtacaa cgataggctc ctgaaactgc
     4801 tgtgtgtgcc tttcggctac tgtatcccca ggtttaagaa cctgagcatc gaggccctgt
     4861 tcgataggaa cgaggccgcc aagttcgaca aggagaaaaa gaaggtgtcc ggcaattccg
     4921 gatccggacc taagaaaaag aggaaggtgg cggccgctta cccatacgat gttccagatt
     4981 acgcttaggc taggaattca ataaaagatc tttattttca ttagatctgt gtgttggttt
     5041 tttgtgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     5101 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     5161 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     5221 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     5281 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5341 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5401 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5461 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     5521 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5581 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     5641 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5701 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5761 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     5821 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5881 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     5941 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     6001 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6061 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6121 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6181 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6241 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6301 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6361 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6421 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6481 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6541 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6601 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6661 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6721 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6781 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6841 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6901 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6961 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7021 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7081 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7141 caaaggatct tc
//
