LOCUS       sequence-380383-        6998 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 6998)
  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 6998)
  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-EFS-CasRx-T2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   20..149
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        203..414
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             447..467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             477..3374
                     /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             3384..3404
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3414..3440
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3456..3509
                     /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             3510..4217
                     /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(3652..3670)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3920..3939
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4224..4272
                     /note="synthetic polyadenylation signal"
     repeat_region   4280..4420
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4280..4409
                     /label=AAV2 ITR
     rep_origin      4495..4950
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4582..4601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4792..4813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4967..4986)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5086..5108
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5146..5164)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5232..5336
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5337..5405,5406..6197)
                     /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(5555..5574)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6368..6956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6857..6876
                     /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 cctagagtcg
      181 agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
      241 gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt aaactgggaa
      301 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
      361 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg
      421 tgacgcggac cggtgccacc atgagcccca agaagaagag aaaggtggag gccagcatcg
      481 aaaaaaaaaa gtccttcgcc aagggcatgg gcgtgaagtc cacactcgtg tccggctcca
      541 aagtgtacat gacaaccttc gccgaaggca gcgacgccag gctggaaaag atcgtggagg
      601 gcgacagcat caggagcgtg aatgagggcg aggccttcag cgctgaaatg gccgataaaa
      661 acgccggcta taagatcggc aacgccaaat tcagccatcc taagggctac gccgtggtgg
      721 ctaacaaccc tctgtataca ggacccgtcc agcaggatat gctcggcctg aaggaaactc
      781 tggaaaagag gtacttcggc gagagcgctg atggcaatga caatatttgt atccaggtga
      841 tccataacat cctggacatt gaaaaaatcc tcgccgaata cattaccaac gccgcctacg
      901 ccgtcaacaa tatctccggc ctggataagg acattattgg attcggcaag ttctccacag
      961 tgtataccta cgacgaattc aaagaccccg agcaccatag ggccgctttc aacaataacg
     1021 ataagctcat caacgccatc aaggcccagt atgacgagtt cgacaacttc ctcgataacc
     1081 ccagactcgg ctatttcggc caggcctttt tcagcaagga gggcagaaat tacatcatca
     1141 attacggcaa cgaatgctat gacattctgg ccctcctgag cggactgagg cactgggtgg
     1201 tccataacaa cgaagaagag tccaggatct ccaggacctg gctctacaac ctcgataaga
     1261 acctcgacaa cgaatacatc tccaccctca actacctcta cgacaggatc accaatgagc
     1321 tgaccaactc cttctccaag aactccgccg ccaacgtgaa ctatattgcc gaaactctgg
     1381 gaatcaaccc tgccgaattc gccgaacaat atttcagatt cagcattatg aaagagcaga
     1441 aaaacctcgg attcaatatc accaagctca gggaagtgat gctggacagg aaggatatgt
     1501 ccgagatcag gaaaaatcat aaggtgttcg actccatcag gaccaaggtc tacaccatga
     1561 tggactttgt gatttatagg tattacatcg aagaggatgc caaggtggct gccgccaata
     1621 agtccctccc cgataatgag aagtccctga gcgagaagga tatctttgtg attaacctga
     1681 ggggctcctt caacgacgac cagaaggatg ccctctacta cgatgaagct aatagaattt
     1741 ggagaaagct cgaaaatatc atgcacaaca tcaaggaatt taggggaaac aagacaagag
     1801 agtataagaa gaaggacgcc cctagactgc ccagaatcct gcccgctggc cgtgatgttt
     1861 ccgccttcag caaactcatg tatgccctga ccatgttcct ggatggcaag gagatcaacg
     1921 acctcctgac caccctgatt aataaattcg ataacatcca gagcttcctg aaggtgatgc
     1981 ctctcatcgg agtcaacgct aagttcgtgg aggaatacgc ctttttcaaa gactccgcca
     2041 agatcgccga tgagctgagg ctgatcaagt ccttcgctag aatgggagaa cctattgccg
     2101 atgccaggag ggccatgtat atcgacgcca tccgtatttt aggaaccaac ctgtcctatg
     2161 atgagctcaa ggccctcgcc gacacctttt ccctggacga gaacggaaac aagctcaaga
     2221 aaggcaagca cggcatgaga aatttcatta ttaataacgt gatcagcaat aaaaggttcc
     2281 actacctgat cagatacggt gatcctgccc acctccatga gatcgccaaa aacgaggccg
     2341 tggtgaagtt cgtgctcggc aggatcgctg acatccagaa aaaacagggc cagaacggca
     2401 agaaccagat cgacaggtac tacgaaactt gtatcggaaa ggataagggc aagagcgtga
     2461 gcgaaaaggt ggacgctctc acaaagatca tcaccggaat gaactacgac caattcgaca
     2521 agaaaaggag cgtcattgag gacaccggca gggaaaacgc cgagagggag aagtttaaaa
     2581 agatcatcag cctgtacctc accgtgatct accacatcct caagaatatt gtcaatatca
     2641 acgccaggta cgtcatcgga ttccattgcg tcgagcgtga tgctcaactg tacaaggaga
     2701 aaggctacga catcaatctc aagaaactgg aagagaaggg attcagctcc gtcaccaagc
     2761 tctgcgctgg cattgatgaa actgcccccg ataagagaaa ggacgtggaa aaggagatgg
     2821 ctgaaagagc caaggagagc attgacagcc tcgagagcgc caaccccaag ctgtatgcca
     2881 attacatcaa atacagcgac gagaagaaag ccgaggagtt caccaggcag attaacaggg
     2941 agaaggccaa aaccgccctg aacgcctacc tgaggaacac caagtggaat gtgatcatca
     3001 gggaggacct cctgagaatt gacaacaaga catgtaccct gttcagaaac aaggccgtcc
     3061 acctggaagt ggccaggtat gtccacgcct atatcaacga cattgccgag gtcaattcct
     3121 acttccaact gtaccattac atcatgcaga gaattatcat gaatgagagg tacgagaaaa
     3181 gcagcggaaa ggtgtccgag tacttcgacg ctgtgaatga cgagaagaag tacaacgata
     3241 ggctcctgaa actgctgtgt gtgcctttcg gctactgtat ccccaggttt aagaacctga
     3301 gcatcgaggc cctgttcgat aggaacgagg ccgccaagtt cgacaaggag aaaaagaagg
     3361 tgtccggcaa ttccggatcc ggacctaaga aaaagaggaa ggtggccgct gcgtacccat
     3421 acgatgttcc agattacgct gccagcggca gtggagaggg cagaggaagt ctgctaacat
     3481 gcggtgacgt cgaggagaat cctggcccag tgagcaaggg cgaggaggat aacatggcca
     3541 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
     3601 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     3661 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
     3721 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     3781 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     3841 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     3901 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     3961 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     4021 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
     4081 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
     4141 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     4201 acgagctgta caagtaaaag cttaataaaa gatctttatt ttcattagat ctgtgtgttg
     4261 gttttttgtg tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4321 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4381 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4441 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4501 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4561 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4621 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4681 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4741 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4801 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     4861 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4921 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4981 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5041 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5101 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5161 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5221 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5281 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5341 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5401 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5461 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5521 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5581 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5641 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5701 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5761 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5821 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5881 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5941 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6001 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg
     6061 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6121 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6181 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6241 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6301 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6361 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6421 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6481 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6541 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6601 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6661 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6721 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6781 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6841 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6901 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6961 ccagcaacgc ggccttttta cggttcctgg ccttttgc
//
