LOCUS       sequence-380344-        7913 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 7913)
  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 7913)
  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-EF1a-CasRx-T2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   25..154
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        167..1345
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          398..1336
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1293..1313
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1367..1387
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1397..4294
                     /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             4304..4324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4334..4360
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4376..4429
                     /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             4430..5137
                     /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(4572..4590)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4840..4859
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5144..5192
                     /note="synthetic polyadenylation signal"
     repeat_region   5200..5340
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5200..5329
                     /label=AAV2 ITR
     rep_origin      5415..5870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5502..5521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5712..5733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5887..5906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6006..6028
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6066..6084)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6152..6256
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6257..6325,6326..7117)
                     /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(6475..6494)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7288..7876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7777..7796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg
       61 ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga
      121 gagggagtgg ccaactccat cactaggggt tcctgcggcc tctagaggct ccggtgcccg
      181 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      241 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      301 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      361 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
      421 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
      481 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
      541 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
      601 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
      661 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
      721 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
      781 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
      841 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
      901 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
      961 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     1021 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     1081 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     1141 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     1201 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     1261 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     1321 ttttcttcca tttcaggtgt cgtgacggac cggtgccacc atgagcccca agaagaagag
     1381 aaaggtggag gccagcatcg aaaaaaaaaa gtccttcgcc aagggcatgg gcgtgaagtc
     1441 cacactcgtg tccggctcca aagtgtacat gacaaccttc gccgaaggca gcgacgccag
     1501 gctggaaaag atcgtggagg gcgacagcat caggagcgtg aatgagggcg aggccttcag
     1561 cgctgaaatg gccgataaaa acgccggcta taagatcggc aacgccaaat tcagccatcc
     1621 taagggctac gccgtggtgg ctaacaaccc tctgtataca ggacccgtcc agcaggatat
     1681 gctcggcctg aaggaaactc tggaaaagag gtacttcggc gagagcgctg atggcaatga
     1741 caatatttgt atccaggtga tccataacat cctggacatt gaaaaaatcc tcgccgaata
     1801 cattaccaac gccgcctacg ccgtcaacaa tatctccggc ctggataagg acattattgg
     1861 attcggcaag ttctccacag tgtataccta cgacgaattc aaagaccccg agcaccatag
     1921 ggccgctttc aacaataacg ataagctcat caacgccatc aaggcccagt atgacgagtt
     1981 cgacaacttc ctcgataacc ccagactcgg ctatttcggc caggcctttt tcagcaagga
     2041 gggcagaaat tacatcatca attacggcaa cgaatgctat gacattctgg ccctcctgag
     2101 cggactgagg cactgggtgg tccataacaa cgaagaagag tccaggatct ccaggacctg
     2161 gctctacaac ctcgataaga acctcgacaa cgaatacatc tccaccctca actacctcta
     2221 cgacaggatc accaatgagc tgaccaactc cttctccaag aactccgccg ccaacgtgaa
     2281 ctatattgcc gaaactctgg gaatcaaccc tgccgaattc gccgaacaat atttcagatt
     2341 cagcattatg aaagagcaga aaaacctcgg attcaatatc accaagctca gggaagtgat
     2401 gctggacagg aaggatatgt ccgagatcag gaaaaatcat aaggtgttcg actccatcag
     2461 gaccaaggtc tacaccatga tggactttgt gatttatagg tattacatcg aagaggatgc
     2521 caaggtggct gccgccaata agtccctccc cgataatgag aagtccctga gcgagaagga
     2581 tatctttgtg attaacctga ggggctcctt caacgacgac cagaaggatg ccctctacta
     2641 cgatgaagct aatagaattt ggagaaagct cgaaaatatc atgcacaaca tcaaggaatt
     2701 taggggaaac aagacaagag agtataagaa gaaggacgcc cctagactgc ccagaatcct
     2761 gcccgctggc cgtgatgttt ccgccttcag caaactcatg tatgccctga ccatgttcct
     2821 ggatggcaag gagatcaacg acctcctgac caccctgatt aataaattcg ataacatcca
     2881 gagcttcctg aaggtgatgc ctctcatcgg agtcaacgct aagttcgtgg aggaatacgc
     2941 ctttttcaaa gactccgcca agatcgccga tgagctgagg ctgatcaagt ccttcgctag
     3001 aatgggagaa cctattgccg atgccaggag ggccatgtat atcgacgcca tccgtatttt
     3061 aggaaccaac ctgtcctatg atgagctcaa ggccctcgcc gacacctttt ccctggacga
     3121 gaacggaaac aagctcaaga aaggcaagca cggcatgaga aatttcatta ttaataacgt
     3181 gatcagcaat aaaaggttcc actacctgat cagatacggt gatcctgccc acctccatga
     3241 gatcgccaaa aacgaggccg tggtgaagtt cgtgctcggc aggatcgctg acatccagaa
     3301 aaaacagggc cagaacggca agaaccagat cgacaggtac tacgaaactt gtatcggaaa
     3361 ggataagggc aagagcgtga gcgaaaaggt ggacgctctc acaaagatca tcaccggaat
     3421 gaactacgac caattcgaca agaaaaggag cgtcattgag gacaccggca gggaaaacgc
     3481 cgagagggag aagtttaaaa agatcatcag cctgtacctc accgtgatct accacatcct
     3541 caagaatatt gtcaatatca acgccaggta cgtcatcgga ttccattgcg tcgagcgtga
     3601 tgctcaactg tacaaggaga aaggctacga catcaatctc aagaaactgg aagagaaggg
     3661 attcagctcc gtcaccaagc tctgcgctgg cattgatgaa actgcccccg ataagagaaa
     3721 ggacgtggaa aaggagatgg ctgaaagagc caaggagagc attgacagcc tcgagagcgc
     3781 caaccccaag ctgtatgcca attacatcaa atacagcgac gagaagaaag ccgaggagtt
     3841 caccaggcag attaacaggg agaaggccaa aaccgccctg aacgcctacc tgaggaacac
     3901 caagtggaat gtgatcatca gggaggacct cctgagaatt gacaacaaga catgtaccct
     3961 gttcagaaac aaggccgtcc acctggaagt ggccaggtat gtccacgcct atatcaacga
     4021 cattgccgag gtcaattcct acttccaact gtaccattac atcatgcaga gaattatcat
     4081 gaatgagagg tacgagaaaa gcagcggaaa ggtgtccgag tacttcgacg ctgtgaatga
     4141 cgagaagaag tacaacgata ggctcctgaa actgctgtgt gtgcctttcg gctactgtat
     4201 ccccaggttt aagaacctga gcatcgaggc cctgttcgat aggaacgagg ccgccaagtt
     4261 cgacaaggag aaaaagaagg tgtccggcaa ttccggatcc ggacctaaga aaaagaggaa
     4321 ggtggccgct gcgtacccat acgatgttcc agattacgct gccagcggca gtggagaggg
     4381 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag tgagcaaggg
     4441 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     4501 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     4561 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     4621 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     4681 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     4741 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     4801 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     4861 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     4921 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     4981 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     5041 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     5101 ccactccacc ggcggcatgg acgagctgta caagtaaaag cttaataaaa gatctttatt
     5161 ttcattagat ctgtgtgttg gttttttgtg tgcggccgca ggaaccccta gtgatggagt
     5221 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     5281 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     5341 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5401 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5461 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     5521 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5581 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5641 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5701 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     5761 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     5821 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     5881 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     5941 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6001 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     6061 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     6121 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6181 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6241 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6301 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6361 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6421 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6481 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6541 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6601 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6661 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6721 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6781 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6841 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6901 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6961 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7021 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7081 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7141 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7201 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7261 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7321 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7381 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7441 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7501 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7561 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7621 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7681 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7741 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7801 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7861 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg cct
//
