LOCUS       sequence-380340-        6214 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 6214)
  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 6214)
  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-CasRx-pA
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   21..150
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        189..400
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             433..453
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             463..3360
                     /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             3370..3390
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3400..3426
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3441..3489
                     /note="synthetic polyadenylation signal"
     repeat_region   3497..3637
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3497..3626
                     /label=AAV2 ITR
     rep_origin      3712..4167
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3799..3818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4009..4030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4184..4203)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4303..4325
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4363..4381)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4449..4553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4554..4622,4623..5414)
                     /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(4772..4791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5585..6173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6074..6093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
       61 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
      121 gagtggccaa ctccatcact aggggttcct gcggcctcta gagtcgagtg gctccggtgc
      181 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      241 caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      301 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      361 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcggaccggt
      421 gccaccatga gccccaagaa gaagagaaag gtggaggcca gcatcgaaaa aaaaaagtcc
      481 ttcgccaagg gcatgggcgt gaagtccaca ctcgtgtccg gctccaaagt gtacatgaca
      541 accttcgccg aaggcagcga cgccaggctg gaaaagatcg tggagggcga cagcatcagg
      601 agcgtgaatg agggcgaggc cttcagcgct gaaatggccg ataaaaacgc cggctataag
      661 atcggcaacg ccaaattcag ccatcctaag ggctacgccg tggtggctaa caaccctctg
      721 tatacaggac ccgtccagca ggatatgctc ggcctgaagg aaactctgga aaagaggtac
      781 ttcggcgaga gcgctgatgg caatgacaat atttgtatcc aggtgatcca taacatcctg
      841 gacattgaaa aaatcctcgc cgaatacatt accaacgccg cctacgccgt caacaatatc
      901 tccggcctgg ataaggacat tattggattc ggcaagttct ccacagtgta tacctacgac
      961 gaattcaaag accccgagca ccatagggcc gctttcaaca ataacgataa gctcatcaac
     1021 gccatcaagg cccagtatga cgagttcgac aacttcctcg ataaccccag actcggctat
     1081 ttcggccagg cctttttcag caaggagggc agaaattaca tcatcaatta cggcaacgaa
     1141 tgctatgaca ttctggccct cctgagcgga ctgaggcact gggtggtcca taacaacgaa
     1201 gaagagtcca ggatctccag gacctggctc tacaacctcg ataagaacct cgacaacgaa
     1261 tacatctcca ccctcaacta cctctacgac aggatcacca atgagctgac caactccttc
     1321 tccaagaact ccgccgccaa cgtgaactat attgccgaaa ctctgggaat caaccctgcc
     1381 gaattcgccg aacaatattt cagattcagc attatgaaag agcagaaaaa cctcggattc
     1441 aatatcacca agctcaggga agtgatgctg gacaggaagg atatgtccga gatcaggaaa
     1501 aatcataagg tgttcgactc catcaggacc aaggtctaca ccatgatgga ctttgtgatt
     1561 tataggtatt acatcgaaga ggatgccaag gtggctgccg ccaataagtc cctccccgat
     1621 aatgagaagt ccctgagcga gaaggatatc tttgtgatta acctgagggg ctccttcaac
     1681 gacgaccaga aggatgccct ctactacgat gaagctaata gaatttggag aaagctcgaa
     1741 aatatcatgc acaacatcaa ggaatttagg ggaaacaaga caagagagta taagaagaag
     1801 gacgccccta gactgcccag aatcctgccc gctggccgtg atgtttccgc cttcagcaaa
     1861 ctcatgtatg ccctgaccat gttcctggat ggcaaggaga tcaacgacct cctgaccacc
     1921 ctgattaata aattcgataa catccagagc ttcctgaagg tgatgcctct catcggagtc
     1981 aacgctaagt tcgtggagga atacgccttt ttcaaagact ccgccaagat cgccgatgag
     2041 ctgaggctga tcaagtcctt cgctagaatg ggagaaccta ttgccgatgc caggagggcc
     2101 atgtatatcg acgccatccg tattttagga accaacctgt cctatgatga gctcaaggcc
     2161 ctcgccgaca ccttttccct ggacgagaac ggaaacaagc tcaagaaagg caagcacggc
     2221 atgagaaatt tcattattaa taacgtgatc agcaataaaa ggttccacta cctgatcaga
     2281 tacggtgatc ctgcccacct ccatgagatc gccaaaaacg aggccgtggt gaagttcgtg
     2341 ctcggcagga tcgctgacat ccagaaaaaa cagggccaga acggcaagaa ccagatcgac
     2401 aggtactacg aaacttgtat cggaaaggat aagggcaaga gcgtgagcga aaaggtggac
     2461 gctctcacaa agatcatcac cggaatgaac tacgaccaat tcgacaagaa aaggagcgtc
     2521 attgaggaca ccggcaggga aaacgccgag agggagaagt ttaaaaagat catcagcctg
     2581 tacctcaccg tgatctacca catcctcaag aatattgtca atatcaacgc caggtacgtc
     2641 atcggattcc attgcgtcga gcgtgatgct caactgtaca aggagaaagg ctacgacatc
     2701 aatctcaaga aactggaaga gaagggattc agctccgtca ccaagctctg cgctggcatt
     2761 gatgaaactg cccccgataa gagaaaggac gtggaaaagg agatggctga aagagccaag
     2821 gagagcattg acagcctcga gagcgccaac cccaagctgt atgccaatta catcaaatac
     2881 agcgacgaga agaaagccga ggagttcacc aggcagatta acagggagaa ggccaaaacc
     2941 gccctgaacg cctacctgag gaacaccaag tggaatgtga tcatcaggga ggacctcctg
     3001 agaattgaca acaagacatg taccctgttc agaaacaagg ccgtccacct ggaagtggcc
     3061 aggtatgtcc acgcctatat caacgacatt gccgaggtca attcctactt ccaactgtac
     3121 cattacatca tgcagagaat tatcatgaat gagaggtacg agaaaagcag cggaaaggtg
     3181 tccgagtact tcgacgctgt gaatgacgag aagaagtaca acgataggct cctgaaactg
     3241 ctgtgtgtgc ctttcggcta ctgtatcccc aggtttaaga acctgagcat cgaggccctg
     3301 ttcgatagga acgaggccgc caagttcgac aaggagaaaa agaaggtgtc cggcaattcc
     3361 ggatccggac ctaagaaaaa gaggaaggtg gcggccgctt acccatacga tgttccagat
     3421 tacgcttagg ctaggaattc aataaaagat ctttattttc attagatctg tgtgttggtt
     3481 ttttgtgtgc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3541 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3601 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3661 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3721 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3781 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3841 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3901 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3961 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4021 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     4081 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4141 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     4201 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4261 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4321 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4381 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4441 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4501 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4561 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4621 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4681 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4741 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4801 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4861 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4921 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4981 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5041 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5101 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5161 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5221 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     5281 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5341 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5401 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5461 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5521 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5581 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5641 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5701 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5761 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5821 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5881 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5941 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6001 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6061 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6121 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6181 gcaacgcggc ctttttacgg ttcctggcct tttg
//
