LOCUS       sequence-380386-        6998 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 6998)
  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 6998)
  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-hfCas13d-T2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      8..596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     497..516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   658..787
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        841..1052
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1085..1105
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1115..4012
                     /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             4022..4042
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4052..4078
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4094..4147
                     /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             4148..4855
                     /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(4290..4308)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4558..4577
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4862..4910
                     /note="synthetic polyadenylation signal"
     repeat_region   4918..5058
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4918..5047
                     /label=AAV2 ITR
     rep_origin      5133..5588
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5220..5239)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5430..5451
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5605..5624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5724..5746
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5784..5802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5870..5974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5975..6043,6044..6835)
                     /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(6193..6212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
       61 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      121 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      181 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      241 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      301 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      361 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
      421 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
      481 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
      541 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
      601 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct
      661 gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt
      721 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
      781 ggttcctgcg gcctctagag ctaggcgtcc tagagtcgag tggctccggt gcccgtcagt
      841 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
      901 cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      961 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     1021 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcggaccg gtgccaccat
     1081 gagccccaag aagaagagaa aggtggaggc cagcatcgaa aaaaaaaagt ccttcgccaa
     1141 gggcatgggc gtgaagtcca cactcgtgtc cggctccaaa gtgtacatga caaccttcgc
     1201 cgaaggcagc gacgccaggc tggaaaagat cgtggagggc gacagcatca ggagcgtgaa
     1261 tgagggcgag gccttcagcg ctgaaatggc cgataaaaac gccggctata agatcggcaa
     1321 cgccaaattc agccatccta agggctacgc cgtggtggct aacaaccctc tgtatacagg
     1381 acccgtccag caggatatgc tcggcctgaa ggaaactctg gaaaagaggt acttcggcga
     1441 gagcgctgat ggcaatgaca atatttgtat ccaggtgatc cataacatcc tggacattga
     1501 aaaaatcctc gtggaataca ttaccaacgt ggtgtacgtg gtcaacaata tctccggcct
     1561 ggataaggac attattggat tcggcaagtt ctccacagtg tatacctacg acgaattcaa
     1621 agaccccgag caccataggg ccgctttcaa caataacgat aagctcatca acgccatcaa
     1681 ggcccagtat gacgagttcg acaacttcct cgataacccc agactcggct atttcggcca
     1741 ggcctttttc agcaaggagg gcagaaatta catcatcaat tacggcaacg aatgctatga
     1801 cattctggcc ctcctgagcg gactgaggca ctgggtggtc cataacaacg aagaagagtc
     1861 caggatctcc aggacctggc tctacaacct cgataagaac ctcgacaacg aatacatctc
     1921 caccctcaac tacctctacg acaggatcac caatgagctg accaactcct tctccaagaa
     1981 ctccgccgcc aacgtgaact atattgccga aactctggga atcaaccctg ccgaattcgc
     2041 cgaacaatat ttcagattca gcattatgaa agagcagaaa aacctcggat tcaatatcac
     2101 caagctcagg gaagtgatgc tggacaggaa ggatatgtcc gagatcagga aaaatcataa
     2161 ggtgttcgac tccatcagga ccaaggtcta caccatgatg gactttgtga tttataggta
     2221 ttacatcgaa gaggatgcca aggtggctgc cgccaataag tccctccccg ataatgagaa
     2281 gtccctgagc gagaaggata tctttgtgat taacctgagg ggctccttca acgacgacca
     2341 gaaggatgcc ctctactacg atgaagctaa tagaatttgg agaaagctcg aaaatatcat
     2401 gcacaacatc aaggaattta ggggaaacaa gacaagagag tataagaaga aggacgcccc
     2461 tagactgccc agaatcctgc ccgctggccg tgatgtttcc gccttcagca aactcatgta
     2521 tgccctgacc atgttcctgg atggcaagga gatcaacgac ctcctgacca ccctgattaa
     2581 taaattcgat aacatccaga gcttcctgaa ggtgatgcct ctcatcggag tcaacgctaa
     2641 gttcgtggag gaatacgcct ttttcaaaga ctccgccaag atcgccgatg agctgaggct
     2701 gatcaagtcc ttcgctagaa tgggagaacc tattgccgat gccaggaggg ccatgtatat
     2761 cgacgccatc cgtattttag gaaccaacct gtcctatgat gagctcaagg ccctcgccga
     2821 caccttttcc ctggacgaga acggaaacaa gctcaagaaa ggcaagcacg gcatgagaaa
     2881 tttcattatt aataacgtga tcagcaataa aaggttccac tacctgatca gatacggtga
     2941 tcctgcccac ctccatgaga tcgccaaaaa cgaggccgtg gtgaagttcg tgctcggcag
     3001 gatcgctgac atccagaaaa aacagggcca gaacggcaag aaccagatcg acaggtacta
     3061 cgaaacttgt atcggaaagg ataagggcaa gagcgtgagc gaaaaggtgg acgctctcac
     3121 aaagatcatc accggaatga actacgacca attcgacaag aaaaggagcg tcattgagga
     3181 caccggcagg gaaaacgccg agagggagaa gtttaaaaag atcatcagcc tgtacctcac
     3241 cgtgatctac cacatcctca agaatattgt caatatcaac gccaggtacg tcatcggatt
     3301 ccattgcgtc gagcgtgatg ctcaactgta caaggagaaa ggctacgaca tcaatctcaa
     3361 gaaactggaa gagaagggat tcagctccgt caccaagctc tgcgctggca ttgatgaaac
     3421 tgcccccgat aagagaaagg acgtggaaaa ggagatggct gaaagagcca aggagagcat
     3481 tgacagcctc gagagcgcca accccaagct gtatgccaat tacatcaaat acagcgacga
     3541 gaagaaagcc gaggagttca ccaggcagat taacagggag aaggccaaaa ccgccctgaa
     3601 cgcctacctg aggaacacca agtggaatgt gatcatcagg gaggacctcc tgagaattga
     3661 caacaagaca tgtaccctgt tcagaaacaa ggccgtccac ctggaagtgg ccaggtatgt
     3721 ccacgcctat atcaacgaca ttgccgaggt caattcctac ttccaactgt accattacat
     3781 catgcagaga attatcatga atgagaggta cgagaaaagc agcggaaagg tgtccgagta
     3841 cttcgacgct gtgaatgacg agaagaagta caacgatagg ctcctgaaac tgctgtgtgt
     3901 gcctttcggc tactgtatcc ccaggtttaa gaacctgagc atcgaggccc tgttcgatag
     3961 gaacgaggcc gccaagttcg acaaggagaa aaagaaggtg tccggcaatt ccggatccgg
     4021 acctaagaaa aagaggaagg tggccgctgc gtacccatac gatgttccag attacgctgc
     4081 cagcggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc
     4141 tggcccagtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     4201 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     4261 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     4321 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     4381 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     4441 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     4501 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     4561 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     4621 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     4681 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     4741 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     4801 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaaagct
     4861 taataaaaga tctttatttt cattagatct gtgtgttggt tttttgtgtg cggccgcagg
     4921 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4981 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     5041 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5101 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     5161 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     5221 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5281 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5341 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5401 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5461 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     5521 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5581 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5641 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5701 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5761 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5821 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5881 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5941 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6001 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6061 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6121 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6181 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6241 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6301 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6361 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6421 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6481 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6541 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6601 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6661 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     6721 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6781 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6841 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6901 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6961 gttccactga gcgtcagacc ccgtagaaaa gatcaaag
//
