LOCUS       sequence-381962-        6829 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 6829)
  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 6829)
  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)-0.5Syn-CasRx-pA
FEATURES             Location/Qualifiers
     source          1..6829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   10..139
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        158..406
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     158..178
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     329..348
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        408..437
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             1037..1057
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1067..3964
                     /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             3974..3994
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4004..4030
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4045..4093
                     /note="synthetic polyadenylation signal"
     repeat_region   4101..4241
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4101..4230
                     /label=AAV2 ITR
     rep_origin      4316..4771
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4403..4422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4613..4634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4788..4807)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4907..4929
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4967..4985)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5053..5157
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5158..5226,5227..6018)
                     /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(5376..5395)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6189..6777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6678..6697
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
       61 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
      121 tccatcacta ggggttcctg cggcctctag agctagcgag ggcctatttc ccatgattcc
      181 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
      241 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      301 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      361 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgaac ccctaccaac
      421 tggtcggggt ttgaaacgga agagcgagct cttctttttt ttctgtacaa cgcgtacaag
      481 tatctgggaa gggtaacaat gcaagttagc ctgaggattt aggaggaggc tgaaaaacag
      541 agtaggagcc ttactacggg tccagaccct acggacaaga acccccactc ccactcccca
      601 aattgcgcat tccctccccc atcagagggg gaggggaaga ggatgcagcg cggcgcggcg
      661 cgtgcgcact gtcggattta gtaccgcgga cagagccttc gcccccgctg ccggcgcgcg
      721 ccaccacctc cccagcacca aaggcgggct gacgtcactc tccagccctc cccaaactcc
      781 cctacctcac cgccttggtc gcgtccgtgc agcggtgagt ccagtcgggc cgcaccacaa
      841 gaggtgcaag ataggggggt gcaggcgcga ccatacgctc tgcggcggca gagcctcagc
      901 gctgcctcag tctgcagcgg gcagcagagg agtcgcgtcg tgccagagag cgccgccgtg
      961 ctcctgagcc ccttgcgctc cgcccccgcg gcccaccgac ccactgcccc ttggatccac
     1021 cggtgccacc atgagcccca agaagaagag aaaggtggag gccagcatcg aaaaaaaaaa
     1081 gtccttcgcc aagggcatgg gcgtgaagtc cacactcgtg tccggctcca aagtgtacat
     1141 gacaaccttc gccgaaggca gcgacgccag gctggaaaag atcgtggagg gcgacagcat
     1201 caggagcgtg aatgagggcg aggccttcag cgctgaaatg gccgataaaa acgccggcta
     1261 taagatcggc aacgccaaat tcagccatcc taagggctac gccgtggtgg ctaacaaccc
     1321 tctgtataca ggacccgtcc agcaggatat gctcggcctg aaggaaactc tggaaaagag
     1381 gtacttcggc gagagcgctg atggcaatga caatatttgt atccaggtga tccataacat
     1441 cctggacatt gaaaaaatcc tcgccgaata cattaccaac gccgcctacg ccgtcaacaa
     1501 tatctccggc ctggataagg acattattgg attcggcaag ttctccacag tgtataccta
     1561 cgacgaattc aaagaccccg agcaccatag ggccgctttc aacaataacg ataagctcat
     1621 caacgccatc aaggcccagt atgacgagtt cgacaacttc ctcgataacc ccagactcgg
     1681 ctatttcggc caggcctttt tcagcaagga gggcagaaat tacatcatca attacggcaa
     1741 cgaatgctat gacattctgg ccctcctgag cggactgagg cactgggtgg tccataacaa
     1801 cgaagaagag tccaggatct ccaggacctg gctctacaac ctcgataaga acctcgacaa
     1861 cgaatacatc tccaccctca actacctcta cgacaggatc accaatgagc tgaccaactc
     1921 cttctccaag aactccgccg ccaacgtgaa ctatattgcc gaaactctgg gaatcaaccc
     1981 tgccgaattc gccgaacaat atttcagatt cagcattatg aaagagcaga aaaacctcgg
     2041 attcaatatc accaagctca gggaagtgat gctggacagg aaggatatgt ccgagatcag
     2101 gaaaaatcat aaggtgttcg actccatcag gaccaaggtc tacaccatga tggactttgt
     2161 gatttatagg tattacatcg aagaggatgc caaggtggct gccgccaata agtccctccc
     2221 cgataatgag aagtccctga gcgagaagga tatctttgtg attaacctga ggggctcctt
     2281 caacgacgac cagaaggatg ccctctacta cgatgaagct aatagaattt ggagaaagct
     2341 cgaaaatatc atgcacaaca tcaaggaatt taggggaaac aagacaagag agtataagaa
     2401 gaaggacgcc cctagactgc ccagaatcct gcccgctggc cgtgatgttt ccgccttcag
     2461 caaactcatg tatgccctga ccatgttcct ggatggcaag gagatcaacg acctcctgac
     2521 caccctgatt aataaattcg ataacatcca gagcttcctg aaggtgatgc ctctcatcgg
     2581 agtcaacgct aagttcgtgg aggaatacgc ctttttcaaa gactccgcca agatcgccga
     2641 tgagctgagg ctgatcaagt ccttcgctag aatgggagaa cctattgccg atgccaggag
     2701 ggccatgtat atcgacgcca tccgtatttt aggaaccaac ctgtcctatg atgagctcaa
     2761 ggccctcgcc gacacctttt ccctggacga gaacggaaac aagctcaaga aaggcaagca
     2821 cggcatgaga aatttcatta ttaataacgt gatcagcaat aaaaggttcc actacctgat
     2881 cagatacggt gatcctgccc acctccatga gatcgccaaa aacgaggccg tggtgaagtt
     2941 cgtgctcggc aggatcgctg acatccagaa aaaacagggc cagaacggca agaaccagat
     3001 cgacaggtac tacgaaactt gtatcggaaa ggataagggc aagagcgtga gcgaaaaggt
     3061 ggacgctctc acaaagatca tcaccggaat gaactacgac caattcgaca agaaaaggag
     3121 cgtcattgag gacaccggca gggaaaacgc cgagagggag aagtttaaaa agatcatcag
     3181 cctgtacctc accgtgatct accacatcct caagaatatt gtcaatatca acgccaggta
     3241 cgtcatcgga ttccattgcg tcgagcgtga tgctcaactg tacaaggaga aaggctacga
     3301 catcaatctc aagaaactgg aagagaaggg attcagctcc gtcaccaagc tctgcgctgg
     3361 cattgatgaa actgcccccg ataagagaaa ggacgtggaa aaggagatgg ctgaaagagc
     3421 caaggagagc attgacagcc tcgagagcgc caaccccaag ctgtatgcca attacatcaa
     3481 atacagcgac gagaagaaag ccgaggagtt caccaggcag attaacaggg agaaggccaa
     3541 aaccgccctg aacgcctacc tgaggaacac caagtggaat gtgatcatca gggaggacct
     3601 cctgagaatt gacaacaaga catgtaccct gttcagaaac aaggccgtcc acctggaagt
     3661 ggccaggtat gtccacgcct atatcaacga cattgccgag gtcaattcct acttccaact
     3721 gtaccattac atcatgcaga gaattatcat gaatgagagg tacgagaaaa gcagcggaaa
     3781 ggtgtccgag tacttcgacg ctgtgaatga cgagaagaag tacaacgata ggctcctgaa
     3841 actgctgtgt gtgcctttcg gctactgtat ccccaggttt aagaacctga gcatcgaggc
     3901 cctgttcgat aggaacgagg ccgccaagtt cgacaaggag aaaaagaagg tgtccggcaa
     3961 ttccggatcc ggacctaaga aaaagaggaa ggtggcggcc gcttacccat acgatgttcc
     4021 agattacgct taggctagga attcaataaa agatctttat tttcattaga tctgtgtgtt
     4081 ggttttttgt gtgcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4141 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4201 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4261 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4321 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4381 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4441 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4501 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4561 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4621 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     4681 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4741 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4801 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4861 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4921 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4981 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5041 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5101 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5161 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5221 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5281 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5341 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5401 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5461 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5521 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5581 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5641 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5701 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5761 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5821 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     5881 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5941 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6001 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6061 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6121 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6181 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6241 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6301 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6361 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6421 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6481 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6541 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6601 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6661 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6721 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6781 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttg
//
