LOCUS       sequence-380375-        6544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express CasRX and a gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6544)
  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 6544)
  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-U6-DR30(SapI)-EFS-CasRx-pA
FEATURES             Location/Qualifiers
     source          1..6544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        395..424
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        495..706
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             739..759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             769..3666
                     /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             3676..3696
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3706..3732
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3747..3795
                     /note="synthetic polyadenylation signal"
     repeat_region   3803..3943
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3803..3932
                     /label=AAV2 ITR
     rep_origin      4018..4473
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4105..4124)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4315..4336
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4490..4509)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4609..4631
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4669..4687)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4755..4859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4860..4928,4929..5720)
                     /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(5078..5097)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5891..6479
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6380..6399
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6541..6544,1..126)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc
       61 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
      121 gttcctgcgg cctctagagc tagcgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgaacccc taccaactgg tcggggtttg
      421 aaacggaaga gcgagctctt cttttttttc tgtacaacgc gtcctagagt cgagtggctc
      481 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      541 ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      601 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
      661 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg
      721 accggtgcca ccatgagccc caagaagaag agaaaggtgg aggccagcat cgaaaaaaaa
      781 aagtccttcg ccaagggcat gggcgtgaag tccacactcg tgtccggctc caaagtgtac
      841 atgacaacct tcgccgaagg cagcgacgcc aggctggaaa agatcgtgga gggcgacagc
      901 atcaggagcg tgaatgaggg cgaggccttc agcgctgaaa tggccgataa aaacgccggc
      961 tataagatcg gcaacgccaa attcagccat cctaagggct acgccgtggt ggctaacaac
     1021 cctctgtata caggacccgt ccagcaggat atgctcggcc tgaaggaaac tctggaaaag
     1081 aggtacttcg gcgagagcgc tgatggcaat gacaatattt gtatccaggt gatccataac
     1141 atcctggaca ttgaaaaaat cctcgccgaa tacattacca acgccgccta cgccgtcaac
     1201 aatatctccg gcctggataa ggacattatt ggattcggca agttctccac agtgtatacc
     1261 tacgacgaat tcaaagaccc cgagcaccat agggccgctt tcaacaataa cgataagctc
     1321 atcaacgcca tcaaggccca gtatgacgag ttcgacaact tcctcgataa ccccagactc
     1381 ggctatttcg gccaggcctt tttcagcaag gagggcagaa attacatcat caattacggc
     1441 aacgaatgct atgacattct ggccctcctg agcggactga ggcactgggt ggtccataac
     1501 aacgaagaag agtccaggat ctccaggacc tggctctaca acctcgataa gaacctcgac
     1561 aacgaataca tctccaccct caactacctc tacgacagga tcaccaatga gctgaccaac
     1621 tccttctcca agaactccgc cgccaacgtg aactatattg ccgaaactct gggaatcaac
     1681 cctgccgaat tcgccgaaca atatttcaga ttcagcatta tgaaagagca gaaaaacctc
     1741 ggattcaata tcaccaagct cagggaagtg atgctggaca ggaaggatat gtccgagatc
     1801 aggaaaaatc ataaggtgtt cgactccatc aggaccaagg tctacaccat gatggacttt
     1861 gtgatttata ggtattacat cgaagaggat gccaaggtgg ctgccgccaa taagtccctc
     1921 cccgataatg agaagtccct gagcgagaag gatatctttg tgattaacct gaggggctcc
     1981 ttcaacgacg accagaagga tgccctctac tacgatgaag ctaatagaat ttggagaaag
     2041 ctcgaaaata tcatgcacaa catcaaggaa tttaggggaa acaagacaag agagtataag
     2101 aagaaggacg cccctagact gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc
     2161 agcaaactca tgtatgccct gaccatgttc ctggatggca aggagatcaa cgacctcctg
     2221 accaccctga ttaataaatt cgataacatc cagagcttcc tgaaggtgat gcctctcatc
     2281 ggagtcaacg ctaagttcgt ggaggaatac gcctttttca aagactccgc caagatcgcc
     2341 gatgagctga ggctgatcaa gtccttcgct agaatgggag aacctattgc cgatgccagg
     2401 agggccatgt atatcgacgc catccgtatt ttaggaacca acctgtccta tgatgagctc
     2461 aaggccctcg ccgacacctt ttccctggac gagaacggaa acaagctcaa gaaaggcaag
     2521 cacggcatga gaaatttcat tattaataac gtgatcagca ataaaaggtt ccactacctg
     2581 atcagatacg gtgatcctgc ccacctccat gagatcgcca aaaacgaggc cgtggtgaag
     2641 ttcgtgctcg gcaggatcgc tgacatccag aaaaaacagg gccagaacgg caagaaccag
     2701 atcgacaggt actacgaaac ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag
     2761 gtggacgctc tcacaaagat catcaccgga atgaactacg accaattcga caagaaaagg
     2821 agcgtcattg aggacaccgg cagggaaaac gccgagaggg agaagtttaa aaagatcatc
     2881 agcctgtacc tcaccgtgat ctaccacatc ctcaagaata ttgtcaatat caacgccagg
     2941 tacgtcatcg gattccattg cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac
     3001 gacatcaatc tcaagaaact ggaagagaag ggattcagct ccgtcaccaa gctctgcgct
     3061 ggcattgatg aaactgcccc cgataagaga aaggacgtgg aaaaggagat ggctgaaaga
     3121 gccaaggaga gcattgacag cctcgagagc gccaacccca agctgtatgc caattacatc
     3181 aaatacagcg acgagaagaa agccgaggag ttcaccaggc agattaacag ggagaaggcc
     3241 aaaaccgccc tgaacgccta cctgaggaac accaagtgga atgtgatcat cagggaggac
     3301 ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa acaaggccgt ccacctggaa
     3361 gtggccaggt atgtccacgc ctatatcaac gacattgccg aggtcaattc ctacttccaa
     3421 ctgtaccatt acatcatgca gagaattatc atgaatgaga ggtacgagaa aagcagcgga
     3481 aaggtgtccg agtacttcga cgctgtgaat gacgagaaga agtacaacga taggctcctg
     3541 aaactgctgt gtgtgccttt cggctactgt atccccaggt ttaagaacct gagcatcgag
     3601 gccctgttcg ataggaacga ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc
     3661 aattccggat ccggacctaa gaaaaagagg aaggtggcgg ccgcttaccc atacgatgtt
     3721 ccagattacg cttaggctag gaattcaata aaagatcttt attttcatta gatctgtgtg
     3781 ttggtttttt gtgtgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     3841 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     3901 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     3961 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4021 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4081 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4141 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4201 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4261 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4321 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4381 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4441 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4501 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4561 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4621 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4681 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4741 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4801 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4861 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4921 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4981 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5041 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5101 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5161 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5221 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5281 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5341 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5401 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5461 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5521 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     5581 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5641 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5701 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5761 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5821 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5881 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5941 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6001 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6061 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6121 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6181 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6241 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6301 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6361 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6421 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6481 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6541 cctg
//
