LOCUS       sequence-380342-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express CasRx  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  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 7104)
  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-0.4CaMKII-CasRx-T2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   20..149
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             553..573
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             583..3480
                     /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             3490..3510
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3520..3546
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3562..3615
                     /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             3616..4323
                     /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(3758..3776)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4026..4045
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4330..4378
                     /note="synthetic polyadenylation signal"
     repeat_region   4386..4526
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4386..4515
                     /label=AAV2 ITR
     rep_origin      4601..5056
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4688..4707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4898..4919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5073..5092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5192..5214
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5252..5270)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5338..5442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5443..5511,5512..6303)
                     /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(5661..5680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6474..7062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6963..6982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc
       61 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      121 agtggccaac tccatcacta ggggttcctg cggcctctag agctaggcgt acttgtggac
      181 taagtttgtt cacatcccct tctccaaccc cctcagtaca tcaccctggg agaacaaggt
      241 ccacttgctt ctgggcccac acagtcctgc agtattgtgt atataaggcc agggcaacgg
      301 aggagcaggt tttgaagtga aaggcaggca ggtgttgggg aggcagttac cggggcaacg
      361 ggaacagggc gtttcggagg tggttgccat ggggacctgg atgctgacga aggctcgcga
      421 ggctgtgagc agccacagtg ccctgctcag aagccccaag ctcgtcaatc aagctggttc
      481 tccatttgca ctcaggagca cgggcaggcg agtggcccct agttctggga gcagaccggt
      541 gccaccatga gccccaagaa gaagagaaag gtggaggcca gcatcgaaaa aaaaaagtcc
      601 ttcgccaagg gcatgggcgt gaagtccaca ctcgtgtccg gctccaaagt gtacatgaca
      661 accttcgccg aaggcagcga cgccaggctg gaaaagatcg tggagggcga cagcatcagg
      721 agcgtgaatg agggcgaggc cttcagcgct gaaatggccg ataaaaacgc cggctataag
      781 atcggcaacg ccaaattcag ccatcctaag ggctacgccg tggtggctaa caaccctctg
      841 tatacaggac ccgtccagca ggatatgctc ggcctgaagg aaactctgga aaagaggtac
      901 ttcggcgaga gcgctgatgg caatgacaat atttgtatcc aggtgatcca taacatcctg
      961 gacattgaaa aaatcctcgc cgaatacatt accaacgccg cctacgccgt caacaatatc
     1021 tccggcctgg ataaggacat tattggattc ggcaagttct ccacagtgta tacctacgac
     1081 gaattcaaag accccgagca ccatagggcc gctttcaaca ataacgataa gctcatcaac
     1141 gccatcaagg cccagtatga cgagttcgac aacttcctcg ataaccccag actcggctat
     1201 ttcggccagg cctttttcag caaggagggc agaaattaca tcatcaatta cggcaacgaa
     1261 tgctatgaca ttctggccct cctgagcgga ctgaggcact gggtggtcca taacaacgaa
     1321 gaagagtcca ggatctccag gacctggctc tacaacctcg ataagaacct cgacaacgaa
     1381 tacatctcca ccctcaacta cctctacgac aggatcacca atgagctgac caactccttc
     1441 tccaagaact ccgccgccaa cgtgaactat attgccgaaa ctctgggaat caaccctgcc
     1501 gaattcgccg aacaatattt cagattcagc attatgaaag agcagaaaaa cctcggattc
     1561 aatatcacca agctcaggga agtgatgctg gacaggaagg atatgtccga gatcaggaaa
     1621 aatcataagg tgttcgactc catcaggacc aaggtctaca ccatgatgga ctttgtgatt
     1681 tataggtatt acatcgaaga ggatgccaag gtggctgccg ccaataagtc cctccccgat
     1741 aatgagaagt ccctgagcga gaaggatatc tttgtgatta acctgagggg ctccttcaac
     1801 gacgaccaga aggatgccct ctactacgat gaagctaata gaatttggag aaagctcgaa
     1861 aatatcatgc acaacatcaa ggaatttagg ggaaacaaga caagagagta taagaagaag
     1921 gacgccccta gactgcccag aatcctgccc gctggccgtg atgtttccgc cttcagcaaa
     1981 ctcatgtatg ccctgaccat gttcctggat ggcaaggaga tcaacgacct cctgaccacc
     2041 ctgattaata aattcgataa catccagagc ttcctgaagg tgatgcctct catcggagtc
     2101 aacgctaagt tcgtggagga atacgccttt ttcaaagact ccgccaagat cgccgatgag
     2161 ctgaggctga tcaagtcctt cgctagaatg ggagaaccta ttgccgatgc caggagggcc
     2221 atgtatatcg acgccatccg tattttagga accaacctgt cctatgatga gctcaaggcc
     2281 ctcgccgaca ccttttccct ggacgagaac ggaaacaagc tcaagaaagg caagcacggc
     2341 atgagaaatt tcattattaa taacgtgatc agcaataaaa ggttccacta cctgatcaga
     2401 tacggtgatc ctgcccacct ccatgagatc gccaaaaacg aggccgtggt gaagttcgtg
     2461 ctcggcagga tcgctgacat ccagaaaaaa cagggccaga acggcaagaa ccagatcgac
     2521 aggtactacg aaacttgtat cggaaaggat aagggcaaga gcgtgagcga aaaggtggac
     2581 gctctcacaa agatcatcac cggaatgaac tacgaccaat tcgacaagaa aaggagcgtc
     2641 attgaggaca ccggcaggga aaacgccgag agggagaagt ttaaaaagat catcagcctg
     2701 tacctcaccg tgatctacca catcctcaag aatattgtca atatcaacgc caggtacgtc
     2761 atcggattcc attgcgtcga gcgtgatgct caactgtaca aggagaaagg ctacgacatc
     2821 aatctcaaga aactggaaga gaagggattc agctccgtca ccaagctctg cgctggcatt
     2881 gatgaaactg cccccgataa gagaaaggac gtggaaaagg agatggctga aagagccaag
     2941 gagagcattg acagcctcga gagcgccaac cccaagctgt atgccaatta catcaaatac
     3001 agcgacgaga agaaagccga ggagttcacc aggcagatta acagggagaa ggccaaaacc
     3061 gccctgaacg cctacctgag gaacaccaag tggaatgtga tcatcaggga ggacctcctg
     3121 agaattgaca acaagacatg taccctgttc agaaacaagg ccgtccacct ggaagtggcc
     3181 aggtatgtcc acgcctatat caacgacatt gccgaggtca attcctactt ccaactgtac
     3241 cattacatca tgcagagaat tatcatgaat gagaggtacg agaaaagcag cggaaaggtg
     3301 tccgagtact tcgacgctgt gaatgacgag aagaagtaca acgataggct cctgaaactg
     3361 ctgtgtgtgc ctttcggcta ctgtatcccc aggtttaaga acctgagcat cgaggccctg
     3421 ttcgatagga acgaggccgc caagttcgac aaggagaaaa agaaggtgtc cggcaattcc
     3481 ggatccggac ctaagaaaaa gaggaaggtg gccgctgcgt acccatacga tgttccagat
     3541 tacgctgcca gcggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag
     3601 gagaatcctg gcccagtgag caagggcgag gaggataaca tggccatcat caaggagttc
     3661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     3721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     3781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     3841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     3901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     3961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     4021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     4081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     4141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     4201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     4261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     4321 taaaagctta ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgcg
     4381 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4441 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4501 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4561 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4621 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     4681 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4741 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4801 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4861 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4921 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4981 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5041 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5101 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5161 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5221 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5281 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5341 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5401 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5461 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5521 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5581 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5641 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5701 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5761 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5821 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5881 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5941 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6001 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6061 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6121 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     6181 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6241 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6301 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6361 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6421 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6481 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6541 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6601 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6661 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6721 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6781 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6841 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6901 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6961 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7021 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7081 tttttacggt tcctggcctt ttgc
//
