LOCUS       sequence-467423-        7239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a RfxCas13d-compatible gRNA and to express HA Tagged 
            CasRx from a CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7239)
  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 7239)
  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-DR(RfxCas13d)-CAG-CasRx-pa
FEATURES             Location/Qualifiers
     source          1..7239
                     /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..1043
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     795..815
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     966..985
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1045..1074
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        1174..1477
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1821..2048
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2079..2099
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2109..5006
                     /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             5016..5036
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5046..5072
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5087..5135
                     /note="synthetic polyadenylation signal"
     repeat_region   5152..5292
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5152..5281
                     /label=AAV2 ITR
     rep_origin      5367..5822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5454..5473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5664..5685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5839..5858)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5958..5980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6018..6036)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6104..6208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6209..6277,6278..7069)
                     /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(6427..6446)
                     /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 tagcgagggc ctatttccca tgattccttc atatttgcat atacgataca
      841 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      901 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      961 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1021 tcttgtggaa aggacgaaac accgaacccc taccaactgg tcggggtttg aaacggaaga
     1081 gcgagctctt cttttttttc tgtacaacgc gtctagttat taatagtaat caattacggg
     1141 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1201 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1261 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1321 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1381 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1441 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
     1501 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
     1561 attattttgt gcagcgatgg gggcgggggg gggggggggg ggggcgccgg gggggggggg
     1621 gggggggggg gggggggggg gggggggggg gggggggggg ggggcgcgcg ccaggcgggg
     1681 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga
     1741 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
     1801 agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg
     1861 ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg
     1921 ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa gggtttaagg
     1981 gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct gaaatcactt
     2041 tttttcaggt tggaccggtc atcgatgcca ccatgagccc caagaagaag agaaaggtgg
     2101 aggccagcat cgaaaaaaaa aagtccttcg ccaagggcat gggcgtgaag tccacactcg
     2161 tgtccggctc caaagtgtac atgacaacct tcgccgaagg cagcgacgcc aggctggaaa
     2221 agatcgtgga gggcgacagc atcaggagcg tgaatgaggg cgaggccttc agcgctgaaa
     2281 tggccgataa aaacgccggc tataagatcg gcaacgccaa attcagccat cctaagggct
     2341 acgccgtggt ggctaacaac cctctgtata caggacccgt ccagcaggat atgctcggcc
     2401 tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc tgatggcaat gacaatattt
     2461 gtatccaggt gatccataac atcctggaca ttgaaaaaat cctcgccgaa tacattacca
     2521 acgccgccta cgccgtcaac aatatctccg gcctggataa ggacattatt ggattcggca
     2581 agttctccac agtgtatacc tacgacgaat tcaaagaccc cgagcaccat agggccgctt
     2641 tcaacaataa cgataagctc atcaacgcca tcaaggccca gtatgacgag ttcgacaact
     2701 tcctcgataa ccccagactc ggctatttcg gccaggcctt tttcagcaag gagggcagaa
     2761 attacatcat caattacggc aacgaatgct atgacattct ggccctcctg agcggactga
     2821 ggcactgggt ggtccataac aacgaagaag agtccaggat ctccaggacc tggctctaca
     2881 acctcgataa gaacctcgac aacgaataca tctccaccct caactacctc tacgacagga
     2941 tcaccaatga gctgaccaac tccttctcca agaactccgc cgccaacgtg aactatattg
     3001 ccgaaactct gggaatcaac cctgccgaat tcgccgaaca atatttcaga ttcagcatta
     3061 tgaaagagca gaaaaacctc ggattcaata tcaccaagct cagggaagtg atgctggaca
     3121 ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt cgactccatc aggaccaagg
     3181 tctacaccat gatggacttt gtgatttata ggtattacat cgaagaggat gccaaggtgg
     3241 ctgccgccaa taagtccctc cccgataatg agaagtccct gagcgagaag gatatctttg
     3301 tgattaacct gaggggctcc ttcaacgacg accagaagga tgccctctac tacgatgaag
     3361 ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa catcaaggaa tttaggggaa
     3421 acaagacaag agagtataag aagaaggacg cccctagact gcccagaatc ctgcccgctg
     3481 gccgtgatgt ttccgccttc agcaaactca tgtatgccct gaccatgttc ctggatggca
     3541 aggagatcaa cgacctcctg accaccctga ttaataaatt cgataacatc cagagcttcc
     3601 tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt ggaggaatac gcctttttca
     3661 aagactccgc caagatcgcc gatgagctga ggctgatcaa gtccttcgct agaatgggag
     3721 aacctattgc cgatgccagg agggccatgt atatcgacgc catccgtatt ttaggaacca
     3781 acctgtccta tgatgagctc aaggccctcg ccgacacctt ttccctggac gagaacggaa
     3841 acaagctcaa gaaaggcaag cacggcatga gaaatttcat tattaataac gtgatcagca
     3901 ataaaaggtt ccactacctg atcagatacg gtgatcctgc ccacctccat gagatcgcca
     3961 aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc tgacatccag aaaaaacagg
     4021 gccagaacgg caagaaccag atcgacaggt actacgaaac ttgtatcgga aaggataagg
     4081 gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat catcaccgga atgaactacg
     4141 accaattcga caagaaaagg agcgtcattg aggacaccgg cagggaaaac gccgagaggg
     4201 agaagtttaa aaagatcatc agcctgtacc tcaccgtgat ctaccacatc ctcaagaata
     4261 ttgtcaatat caacgccagg tacgtcatcg gattccattg cgtcgagcgt gatgctcaac
     4321 tgtacaagga gaaaggctac gacatcaatc tcaagaaact ggaagagaag ggattcagct
     4381 ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc cgataagaga aaggacgtgg
     4441 aaaaggagat ggctgaaaga gccaaggaga gcattgacag cctcgagagc gccaacccca
     4501 agctgtatgc caattacatc aaatacagcg acgagaagaa agccgaggag ttcaccaggc
     4561 agattaacag ggagaaggcc aaaaccgccc tgaacgccta cctgaggaac accaagtgga
     4621 atgtgatcat cagggaggac ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa
     4681 acaaggccgt ccacctggaa gtggccaggt atgtccacgc ctatatcaac gacattgccg
     4741 aggtcaattc ctacttccaa ctgtaccatt acatcatgca gagaattatc atgaatgaga
     4801 ggtacgagaa aagcagcgga aaggtgtccg agtacttcga cgctgtgaat gacgagaaga
     4861 agtacaacga taggctcctg aaactgctgt gtgtgccttt cggctactgt atccccaggt
     4921 ttaagaacct gagcatcgag gccctgttcg ataggaacga ggccgccaag ttcgacaagg
     4981 agaaaaagaa ggtgtccggc aattccggat ccggacctaa gaaaaagagg aaggtggcgg
     5041 ccgcttaccc atacgatgtt ccagattacg cttaggctag gaattcaata aaagatcttt
     5101 attttcatta gatctgtgtg ttggtttttt gtgtgcggac cgagcggccg caggaacccc
     5161 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     5221 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     5281 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5341 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5401 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     5461 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5521 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5581 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5641 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5701 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5761 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5821 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5881 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5941 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6001 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     6061 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6421 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6721 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
