LOCUS       sequence-380376-        7598 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 7598)
  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 7598)
  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-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   32..161
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        180..428
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     180..200
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     351..370
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        430..459
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             1059..1079
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1089..3986
                     /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             3996..4016
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4026..4052
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4068..4121
                     /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             4122..4829
                     /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(4264..4282)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4532..4551
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4836..4884
                     /note="synthetic polyadenylation signal"
     repeat_region   4892..5032
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4892..5021
                     /label=AAV2 ITR
     rep_origin      5107..5562
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5194..5213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5404..5425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5579..5598)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5698..5720
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5758..5776)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5844..5948
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5949..6017,6018..6809)
                     /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(6167..6186)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6980..7568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7469..7488
                     /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 agagctagcg
      181 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      241 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      301 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      361 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
      421 gaaacaccga acccctacca actggtcggg gtttgaaacg gaagagcgag ctcttctttt
      481 ttttctgtac aacgcgtaca agtatctggg aagggtaaca atgcaagtta gcctgaggat
      541 ttaggaggag gctgaaaaac agagtaggag ccttactacg ggtccagacc ctacggacaa
      601 gaacccccac tcccactccc caaattgcgc attccctccc ccatcagagg gggaggggaa
      661 gaggatgcag cgcggcgcgg cgcgtgcgca ctgtcggatt tagtaccgcg gacagagcct
      721 tcgcccccgc tgccggcgcg cgccaccacc tccccagcac caaaggcggg ctgacgtcac
      781 tctccagccc tccccaaact cccctacctc accgccttgg tcgcgtccgt gcagcggtga
      841 gtccagtcgg gccgcaccac aagaggtgca agataggggg gtgcaggcgc gaccatacgc
      901 tctgcggcgg cagagcctca gcgctgcctc agtctgcagc gggcagcaga ggagtcgcgt
      961 cgtgccagag agcgccgccg tgctcctgag ccccttgcgc tccgcccccg cggcccaccg
     1021 acccactgcc ccttggatcc accggtgcca ccatgagccc caagaagaag agaaaggtgg
     1081 aggccagcat cgaaaaaaaa aagtccttcg ccaagggcat gggcgtgaag tccacactcg
     1141 tgtccggctc caaagtgtac atgacaacct tcgccgaagg cagcgacgcc aggctggaaa
     1201 agatcgtgga gggcgacagc atcaggagcg tgaatgaggg cgaggccttc agcgctgaaa
     1261 tggccgataa aaacgccggc tataagatcg gcaacgccaa attcagccat cctaagggct
     1321 acgccgtggt ggctaacaac cctctgtata caggacccgt ccagcaggat atgctcggcc
     1381 tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc tgatggcaat gacaatattt
     1441 gtatccaggt gatccataac atcctggaca ttgaaaaaat cctcgccgaa tacattacca
     1501 acgccgccta cgccgtcaac aatatctccg gcctggataa ggacattatt ggattcggca
     1561 agttctccac agtgtatacc tacgacgaat tcaaagaccc cgagcaccat agggccgctt
     1621 tcaacaataa cgataagctc atcaacgcca tcaaggccca gtatgacgag ttcgacaact
     1681 tcctcgataa ccccagactc ggctatttcg gccaggcctt tttcagcaag gagggcagaa
     1741 attacatcat caattacggc aacgaatgct atgacattct ggccctcctg agcggactga
     1801 ggcactgggt ggtccataac aacgaagaag agtccaggat ctccaggacc tggctctaca
     1861 acctcgataa gaacctcgac aacgaataca tctccaccct caactacctc tacgacagga
     1921 tcaccaatga gctgaccaac tccttctcca agaactccgc cgccaacgtg aactatattg
     1981 ccgaaactct gggaatcaac cctgccgaat tcgccgaaca atatttcaga ttcagcatta
     2041 tgaaagagca gaaaaacctc ggattcaata tcaccaagct cagggaagtg atgctggaca
     2101 ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt cgactccatc aggaccaagg
     2161 tctacaccat gatggacttt gtgatttata ggtattacat cgaagaggat gccaaggtgg
     2221 ctgccgccaa taagtccctc cccgataatg agaagtccct gagcgagaag gatatctttg
     2281 tgattaacct gaggggctcc ttcaacgacg accagaagga tgccctctac tacgatgaag
     2341 ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa catcaaggaa tttaggggaa
     2401 acaagacaag agagtataag aagaaggacg cccctagact gcccagaatc ctgcccgctg
     2461 gccgtgatgt ttccgccttc agcaaactca tgtatgccct gaccatgttc ctggatggca
     2521 aggagatcaa cgacctcctg accaccctga ttaataaatt cgataacatc cagagcttcc
     2581 tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt ggaggaatac gcctttttca
     2641 aagactccgc caagatcgcc gatgagctga ggctgatcaa gtccttcgct agaatgggag
     2701 aacctattgc cgatgccagg agggccatgt atatcgacgc catccgtatt ttaggaacca
     2761 acctgtccta tgatgagctc aaggccctcg ccgacacctt ttccctggac gagaacggaa
     2821 acaagctcaa gaaaggcaag cacggcatga gaaatttcat tattaataac gtgatcagca
     2881 ataaaaggtt ccactacctg atcagatacg gtgatcctgc ccacctccat gagatcgcca
     2941 aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc tgacatccag aaaaaacagg
     3001 gccagaacgg caagaaccag atcgacaggt actacgaaac ttgtatcgga aaggataagg
     3061 gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat catcaccgga atgaactacg
     3121 accaattcga caagaaaagg agcgtcattg aggacaccgg cagggaaaac gccgagaggg
     3181 agaagtttaa aaagatcatc agcctgtacc tcaccgtgat ctaccacatc ctcaagaata
     3241 ttgtcaatat caacgccagg tacgtcatcg gattccattg cgtcgagcgt gatgctcaac
     3301 tgtacaagga gaaaggctac gacatcaatc tcaagaaact ggaagagaag ggattcagct
     3361 ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc cgataagaga aaggacgtgg
     3421 aaaaggagat ggctgaaaga gccaaggaga gcattgacag cctcgagagc gccaacccca
     3481 agctgtatgc caattacatc aaatacagcg acgagaagaa agccgaggag ttcaccaggc
     3541 agattaacag ggagaaggcc aaaaccgccc tgaacgccta cctgaggaac accaagtgga
     3601 atgtgatcat cagggaggac ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa
     3661 acaaggccgt ccacctggaa gtggccaggt atgtccacgc ctatatcaac gacattgccg
     3721 aggtcaattc ctacttccaa ctgtaccatt acatcatgca gagaattatc atgaatgaga
     3781 ggtacgagaa aagcagcgga aaggtgtccg agtacttcga cgctgtgaat gacgagaaga
     3841 agtacaacga taggctcctg aaactgctgt gtgtgccttt cggctactgt atccccaggt
     3901 ttaagaacct gagcatcgag gccctgttcg ataggaacga ggccgccaag ttcgacaagg
     3961 agaaaaagaa ggtgtccggc aattccggat ccggacctaa gaaaaagagg aaggtggccg
     4021 ctgcgtaccc atacgatgtt ccagattacg ctgccagcgg cagtggagag ggcagaggaa
     4081 gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagg
     4141 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     4201 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     4261 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     4321 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     4381 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     4441 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     4501 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     4561 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     4621 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     4681 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     4741 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     4801 ccggcggcat ggacgagctg tacaagtaaa agcttaataa aagatcttta ttttcattag
     4861 atctgtgtgt tggttttttg tgtgcggccg caggaacccc tagtgatgga gttggccact
     4921 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4981 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     5041 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     5101 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5161 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     5221 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5281 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     5341 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5401 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     5461 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     5521 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     5581 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     5641 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5701 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     5761 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5821 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5881 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5941 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6001 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6061 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6121 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6181 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6241 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6301 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6361 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6421 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6481 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6541 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6601 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6661 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6721 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6781 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6841 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6901 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     7141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7561 tatggaaaaa cgccagcaac gcggcctttt tacggttc
//
