LOCUS       sequence-380339-        7313 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 7313)
  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 7313)
  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-T2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7313
                     /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        169..417
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     169..189
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     340..359
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..448
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        519..730
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             763..783
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             793..3690
                     /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             3700..3720
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3730..3756
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3772..3825
                     /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             3826..4533
                     /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(3968..3986)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4236..4255
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4540..4588
                     /note="synthetic polyadenylation signal"
     repeat_region   4596..4736
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4596..4725
                     /label=AAV2 ITR
     rep_origin      4811..5266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4898..4917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5108..5129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5283..5302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5402..5424
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5462..5480)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5548..5652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5653..5721,5722..6513)
                     /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(5871..5890)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6684..7272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7173..7192
                     /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 gagctagcga gggcctattt
      181 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
      241 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      301 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      361 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgaa
      421 cccctaccaa ctggtcgggg tttgaaacgg aagagcgagc tcttcttttt tttctgtaca
      481 acgcgtccta gagtcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
      541 agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg
      601 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
      661 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
      721 cagaacacag gtgtcgtgac gcggaccggt gccaccatga gccccaagaa gaagagaaag
      781 gtggaggcca gcatcgaaaa aaaaaagtcc ttcgccaagg gcatgggcgt gaagtccaca
      841 ctcgtgtccg gctccaaagt gtacatgaca accttcgccg aaggcagcga cgccaggctg
      901 gaaaagatcg tggagggcga cagcatcagg agcgtgaatg agggcgaggc cttcagcgct
      961 gaaatggccg ataaaaacgc cggctataag atcggcaacg ccaaattcag ccatcctaag
     1021 ggctacgccg tggtggctaa caaccctctg tatacaggac ccgtccagca ggatatgctc
     1081 ggcctgaagg aaactctgga aaagaggtac ttcggcgaga gcgctgatgg caatgacaat
     1141 atttgtatcc aggtgatcca taacatcctg gacattgaaa aaatcctcgc cgaatacatt
     1201 accaacgccg cctacgccgt caacaatatc tccggcctgg ataaggacat tattggattc
     1261 ggcaagttct ccacagtgta tacctacgac gaattcaaag accccgagca ccatagggcc
     1321 gctttcaaca ataacgataa gctcatcaac gccatcaagg cccagtatga cgagttcgac
     1381 aacttcctcg ataaccccag actcggctat ttcggccagg cctttttcag caaggagggc
     1441 agaaattaca tcatcaatta cggcaacgaa tgctatgaca ttctggccct cctgagcgga
     1501 ctgaggcact gggtggtcca taacaacgaa gaagagtcca ggatctccag gacctggctc
     1561 tacaacctcg ataagaacct cgacaacgaa tacatctcca ccctcaacta cctctacgac
     1621 aggatcacca atgagctgac caactccttc tccaagaact ccgccgccaa cgtgaactat
     1681 attgccgaaa ctctgggaat caaccctgcc gaattcgccg aacaatattt cagattcagc
     1741 attatgaaag agcagaaaaa cctcggattc aatatcacca agctcaggga agtgatgctg
     1801 gacaggaagg atatgtccga gatcaggaaa aatcataagg tgttcgactc catcaggacc
     1861 aaggtctaca ccatgatgga ctttgtgatt tataggtatt acatcgaaga ggatgccaag
     1921 gtggctgccg ccaataagtc cctccccgat aatgagaagt ccctgagcga gaaggatatc
     1981 tttgtgatta acctgagggg ctccttcaac gacgaccaga aggatgccct ctactacgat
     2041 gaagctaata gaatttggag aaagctcgaa aatatcatgc acaacatcaa ggaatttagg
     2101 ggaaacaaga caagagagta taagaagaag gacgccccta gactgcccag aatcctgccc
     2161 gctggccgtg atgtttccgc cttcagcaaa ctcatgtatg ccctgaccat gttcctggat
     2221 ggcaaggaga tcaacgacct cctgaccacc ctgattaata aattcgataa catccagagc
     2281 ttcctgaagg tgatgcctct catcggagtc aacgctaagt tcgtggagga atacgccttt
     2341 ttcaaagact ccgccaagat cgccgatgag ctgaggctga tcaagtcctt cgctagaatg
     2401 ggagaaccta ttgccgatgc caggagggcc atgtatatcg acgccatccg tattttagga
     2461 accaacctgt cctatgatga gctcaaggcc ctcgccgaca ccttttccct ggacgagaac
     2521 ggaaacaagc tcaagaaagg caagcacggc atgagaaatt tcattattaa taacgtgatc
     2581 agcaataaaa ggttccacta cctgatcaga tacggtgatc ctgcccacct ccatgagatc
     2641 gccaaaaacg aggccgtggt gaagttcgtg ctcggcagga tcgctgacat ccagaaaaaa
     2701 cagggccaga acggcaagaa ccagatcgac aggtactacg aaacttgtat cggaaaggat
     2761 aagggcaaga gcgtgagcga aaaggtggac gctctcacaa agatcatcac cggaatgaac
     2821 tacgaccaat tcgacaagaa aaggagcgtc attgaggaca ccggcaggga aaacgccgag
     2881 agggagaagt ttaaaaagat catcagcctg tacctcaccg tgatctacca catcctcaag
     2941 aatattgtca atatcaacgc caggtacgtc atcggattcc attgcgtcga gcgtgatgct
     3001 caactgtaca aggagaaagg ctacgacatc aatctcaaga aactggaaga gaagggattc
     3061 agctccgtca ccaagctctg cgctggcatt gatgaaactg cccccgataa gagaaaggac
     3121 gtggaaaagg agatggctga aagagccaag gagagcattg acagcctcga gagcgccaac
     3181 cccaagctgt atgccaatta catcaaatac agcgacgaga agaaagccga ggagttcacc
     3241 aggcagatta acagggagaa ggccaaaacc gccctgaacg cctacctgag gaacaccaag
     3301 tggaatgtga tcatcaggga ggacctcctg agaattgaca acaagacatg taccctgttc
     3361 agaaacaagg ccgtccacct ggaagtggcc aggtatgtcc acgcctatat caacgacatt
     3421 gccgaggtca attcctactt ccaactgtac cattacatca tgcagagaat tatcatgaat
     3481 gagaggtacg agaaaagcag cggaaaggtg tccgagtact tcgacgctgt gaatgacgag
     3541 aagaagtaca acgataggct cctgaaactg ctgtgtgtgc ctttcggcta ctgtatcccc
     3601 aggtttaaga acctgagcat cgaggccctg ttcgatagga acgaggccgc caagttcgac
     3661 aaggagaaaa agaaggtgtc cggcaattcc ggatccggac ctaagaaaaa gaggaaggtg
     3721 gccgctgcgt acccatacga tgttccagat tacgctgcca gcggcagtgg agagggcaga
     3781 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccagtgag caagggcgag
     3841 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     3901 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     3961 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     4021 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     4081 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     4141 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     4201 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     4261 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     4321 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     4381 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     4441 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     4501 tccaccggcg gcatggacga gctgtacaag taaaagctta ataaaagatc tttattttca
     4561 ttagatctgt gtgttggttt tttgtgtgcg gccgcaggaa cccctagtga tggagttggc
     4621 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4681 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4741 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4801 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4861 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4921 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4981 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5041 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5101 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     5161 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     5221 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5281 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5341 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5401 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5461 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5521 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5581 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5641 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5701 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5761 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5821 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5881 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5941 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6001 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6061 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6121 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6181 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6241 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6301 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6361 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6421 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6481 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6541 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6601 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6661 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6721 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6781 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6841 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6901 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6961 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7021 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7081 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7141 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7201 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7261 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttg
//
