LOCUS       sequence-380381-        7283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express CasRx  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  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 7283)
  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-0.5Syn-CasRX-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   32..161
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             744..764
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             774..3671
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3681..3701
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3711..3737
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3753..3806
                     /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             3807..4514
                     /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(3949..3967)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4217..4236
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4521..4569
                     /note="synthetic polyadenylation signal"
     repeat_region   4577..4717
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4577..4706
                     /label=AAV2 ITR
     rep_origin      4792..5247
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4879..4898)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5089..5110
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5264..5283)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5383..5405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5443..5461)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5529..5633
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5634..5702,5703..6494)
                     /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(5852..5871)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6665..7253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7154..7173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc
       61 actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
      121 gcgcagagag ggagtggcca actccatcac taggggttcc tgcggcctct agagctaggc
      181 gtacaagtat ctgggaaggg taacaatgca agttagcctg aggatttagg aggaggctga
      241 aaaacagagt aggagcctta ctacgggtcc agaccctacg gacaagaacc cccactccca
      301 ctccccaaat tgcgcattcc ctcccccatc agagggggag gggaagagga tgcagcgcgg
      361 cgcggcgcgt gcgcactgtc ggatttagta ccgcggacag agccttcgcc cccgctgccg
      421 gcgcgcgcca ccacctcccc agcaccaaag gcgggctgac gtcactctcc agccctcccc
      481 aaactcccct acctcaccgc cttggtcgcg tccgtgcagc ggtgagtcca gtcgggccgc
      541 accacaagag gtgcaagata ggggggtgca ggcgcgacca tacgctctgc ggcggcagag
      601 cctcagcgct gcctcagtct gcagcgggca gcagaggagt cgcgtcgtgc cagagagcgc
      661 cgccgtgctc ctgagcccct tgcgctccgc ccccgcggcc caccgaccca ctgccccttg
      721 gatccaccgg tgccaccatg agccccaaga agaagagaaa ggtggaggcc agcatcgaaa
      781 aaaaaaagtc cttcgccaag ggcatgggcg tgaagtccac actcgtgtcc ggctccaaag
      841 tgtacatgac aaccttcgcc gaaggcagcg acgccaggct ggaaaagatc gtggagggcg
      901 acagcatcag gagcgtgaat gagggcgagg ccttcagcgc tgaaatggcc gataaaaacg
      961 ccggctataa gatcggcaac gccaaattca gccatcctaa gggctacgcc gtggtggcta
     1021 acaaccctct gtatacagga cccgtccagc aggatatgct cggcctgaag gaaactctgg
     1081 aaaagaggta cttcggcgag agcgctgatg gcaatgacaa tatttgtatc caggtgatcc
     1141 ataacatcct ggacattgaa aaaatcctcg ccgaatacat taccaacgcc gcctacgccg
     1201 tcaacaatat ctccggcctg gataaggaca ttattggatt cggcaagttc tccacagtgt
     1261 atacctacga cgaattcaaa gaccccgagc accatagggc cgctttcaac aataacgata
     1321 agctcatcaa cgccatcaag gcccagtatg acgagttcga caacttcctc gataacccca
     1381 gactcggcta tttcggccag gcctttttca gcaaggaggg cagaaattac atcatcaatt
     1441 acggcaacga atgctatgac attctggccc tcctgagcgg actgaggcac tgggtggtcc
     1501 ataacaacga agaagagtcc aggatctcca ggacctggct ctacaacctc gataagaacc
     1561 tcgacaacga atacatctcc accctcaact acctctacga caggatcacc aatgagctga
     1621 ccaactcctt ctccaagaac tccgccgcca acgtgaacta tattgccgaa actctgggaa
     1681 tcaaccctgc cgaattcgcc gaacaatatt tcagattcag cattatgaaa gagcagaaaa
     1741 acctcggatt caatatcacc aagctcaggg aagtgatgct ggacaggaag gatatgtccg
     1801 agatcaggaa aaatcataag gtgttcgact ccatcaggac caaggtctac accatgatgg
     1861 actttgtgat ttataggtat tacatcgaag aggatgccaa ggtggctgcc gccaataagt
     1921 ccctccccga taatgagaag tccctgagcg agaaggatat ctttgtgatt aacctgaggg
     1981 gctccttcaa cgacgaccag aaggatgccc tctactacga tgaagctaat agaatttgga
     2041 gaaagctcga aaatatcatg cacaacatca aggaatttag gggaaacaag acaagagagt
     2101 ataagaagaa ggacgcccct agactgccca gaatcctgcc cgctggccgt gatgtttccg
     2161 ccttcagcaa actcatgtat gccctgacca tgttcctgga tggcaaggag atcaacgacc
     2221 tcctgaccac cctgattaat aaattcgata acatccagag cttcctgaag gtgatgcctc
     2281 tcatcggagt caacgctaag ttcgtggagg aatacgcctt tttcaaagac tccgccaaga
     2341 tcgccgatga gctgaggctg atcaagtcct tcgctagaat gggagaacct attgccgatg
     2401 ccaggagggc catgtatatc gacgccatcc gtattttagg aaccaacctg tcctatgatg
     2461 agctcaaggc cctcgccgac accttttccc tggacgagaa cggaaacaag ctcaagaaag
     2521 gcaagcacgg catgagaaat ttcattatta ataacgtgat cagcaataaa aggttccact
     2581 acctgatcag atacggtgat cctgcccacc tccatgagat cgccaaaaac gaggccgtgg
     2641 tgaagttcgt gctcggcagg atcgctgaca tccagaaaaa acagggccag aacggcaaga
     2701 accagatcga caggtactac gaaacttgta tcggaaagga taagggcaag agcgtgagcg
     2761 aaaaggtgga cgctctcaca aagatcatca ccggaatgaa ctacgaccaa ttcgacaaga
     2821 aaaggagcgt cattgaggac accggcaggg aaaacgccga gagggagaag tttaaaaaga
     2881 tcatcagcct gtacctcacc gtgatctacc acatcctcaa gaatattgtc aatatcaacg
     2941 ccaggtacgt catcggattc cattgcgtcg agcgtgatgc tcaactgtac aaggagaaag
     3001 gctacgacat caatctcaag aaactggaag agaagggatt cagctccgtc accaagctct
     3061 gcgctggcat tgatgaaact gcccccgata agagaaagga cgtggaaaag gagatggctg
     3121 aaagagccaa ggagagcatt gacagcctcg agagcgccaa ccccaagctg tatgccaatt
     3181 acatcaaata cagcgacgag aagaaagccg aggagttcac caggcagatt aacagggaga
     3241 aggccaaaac cgccctgaac gcctacctga ggaacaccaa gtggaatgtg atcatcaggg
     3301 aggacctcct gagaattgac aacaagacat gtaccctgtt cagaaacaag gccgtccacc
     3361 tggaagtggc caggtatgtc cacgcctata tcaacgacat tgccgaggtc aattcctact
     3421 tccaactgta ccattacatc atgcagagaa ttatcatgaa tgagaggtac gagaaaagca
     3481 gcggaaaggt gtccgagtac ttcgacgctg tgaatgacga gaagaagtac aacgataggc
     3541 tcctgaaact gctgtgtgtg cctttcggct actgtatccc caggtttaag aacctgagca
     3601 tcgaggccct gttcgatagg aacgaggccg ccaagttcga caaggagaaa aagaaggtgt
     3661 ccggcaattc cggatccgga cctaagaaaa agaggaaggt ggccgctgcg tacccatacg
     3721 atgttccaga ttacgctgcc agcggcagtg gagagggcag aggaagtctg ctaacatgcg
     3781 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggaggataac atggccatca
     3841 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     3901 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     3961 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     4021 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     4081 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     4141 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     4201 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
     4261 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     4321 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     4381 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     4441 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     4501 agctgtacaa gtaaaagctt aataaaagat ctttattttc attagatctg tgtgttggtt
     4561 ttttgtgtgc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4621 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4681 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     4741 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     4801 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4861 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4921 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4981 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5041 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5101 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     5161 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5221 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5281 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5341 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5401 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5461 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5581 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5641 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5701 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5761 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5821 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5881 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5941 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6001 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6061 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6121 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6181 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6241 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6301 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     6361 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6421 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6481 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6541 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6601 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6781 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7021 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7141 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7201 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7261 gcaacgcggc ctttttacgg ttc
//
