LOCUS       sequence-331087-        6316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for expressing CasRx.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6316)
  AUTHORS   Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS
  TITLE     Methods for in vitro CRISPR/CasRx-Mediated RNA Editing.
  JOURNAL   Front Cell Dev Biol. 2021 Jun 11;9:667879. doi: 
            10.3389/fcell.2021.667879. eCollection 2021.
  PUBMED    34178991
REFERENCE   2  (bases 1 to 6316)
  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: "Front Cell 
            Dev Biol."; date: "2021-06-11"; volume: "9:667879. doi"; pages: " 
            10.3389/fcell.2021.667879. eCollection 2021"
COMMENT     Sequence Label: pM122: pAAV-EFS-CasRx
FEATURES             Location/Qualifiers
     source          1..6316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      465..1053
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     954..973
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1115..1244
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1308..1519
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1539..1559
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1569..4466
                     /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             4476..4496
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4506..4532
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4542..4676
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4579..4598)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4633..4652
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4693..4833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4693..4822
                     /label=AAV2 ITR
     rep_origin      4908..5363
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4995..5014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5205..5226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5380..5399)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5499..5521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5559..5577)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5645..5749
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5750..5818,5819..6316,1..294)
                     /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(5968..5987)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
       61 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      121 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
      181 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
      241 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
      301 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
      361 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
      421 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
      481 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      541 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      601 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      661 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      721 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      781 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
      841 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
      901 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      961 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1021 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1081 ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg
     1141 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     1201 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtaa
     1261 gctttaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     1321 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag
     1381 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     1441 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     1501 gtttgccgcc agaacacagg tctagagcca ccatgagccc caagaagaag agaaaggtgg
     1561 aggccagcat cgaaaaaaaa aagtccttcg ccaagggcat gggcgtgaag tccacactcg
     1621 tgtccggctc caaagtgtac atgacaacct tcgccgaagg cagcgacgcc aggctggaaa
     1681 agatcgtgga gggcgacagc atcaggagcg tgaatgaggg cgaggccttc agcgctgaaa
     1741 tggccgataa aaacgccggc tataagatcg gcaacgccaa attcagccat cctaagggct
     1801 acgccgtggt ggctaacaac cctctgtata caggacccgt ccagcaggat atgctcggcc
     1861 tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc tgatggcaat gacaatattt
     1921 gtatccaggt gatccataac atcctggaca ttgaaaaaat cctcgccgaa tacattacca
     1981 acgccgccta cgccgtcaac aatatctccg gcctggataa ggacattatt ggattcggca
     2041 agttctccac agtgtatacc tacgacgaat tcaaagaccc cgagcaccat agggccgctt
     2101 tcaacaataa cgataagctc atcaacgcca tcaaggccca gtatgacgag ttcgacaact
     2161 tcctcgataa ccccagactc ggctatttcg gccaggcctt tttcagcaag gagggcagaa
     2221 attacatcat caattacggc aacgaatgct atgacattct ggccctcctg agcggactga
     2281 ggcactgggt ggtccataac aacgaagaag agtccaggat ctccaggacc tggctctaca
     2341 acctcgataa gaacctcgac aacgaataca tctccaccct caactacctc tacgacagga
     2401 tcaccaatga gctgaccaac tccttctcca agaactccgc cgccaacgtg aactatattg
     2461 ccgaaactct gggaatcaac cctgccgaat tcgccgaaca atatttcaga ttcagcatta
     2521 tgaaagagca gaaaaacctc ggattcaata tcaccaagct cagggaagtg atgctggaca
     2581 ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt cgactccatc aggaccaagg
     2641 tctacaccat gatggacttt gtgatttata ggtattacat cgaagaggat gccaaggtgg
     2701 ctgccgccaa taagtccctc cccgataatg agaagtccct gagcgagaag gatatctttg
     2761 tgattaacct gaggggctcc ttcaacgacg accagaagga tgccctctac tacgatgaag
     2821 ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa catcaaggaa tttaggggaa
     2881 acaagacaag agagtataag aagaaggacg cccctagact gcccagaatc ctgcccgctg
     2941 gccgtgatgt ttccgccttc agcaaactca tgtatgccct gaccatgttc ctggatggca
     3001 aggagatcaa cgacctcctg accaccctga ttaataaatt cgataacatc cagagcttcc
     3061 tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt ggaggaatac gcctttttca
     3121 aagactccgc caagatcgcc gatgagctga ggctgatcaa gtccttcgct agaatgggag
     3181 aacctattgc cgatgccagg agggccatgt atatcgacgc catccgtatt ttaggaacca
     3241 acctgtccta tgatgagctc aaggccctcg ccgacacctt ttccctggac gagaacggaa
     3301 acaagctcaa gaaaggcaag cacggcatga gaaatttcat tattaataac gtgatcagca
     3361 ataaaaggtt ccactacctg atcagatacg gtgatcctgc ccacctccat gagatcgcca
     3421 aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc tgacatccag aaaaaacagg
     3481 gccagaacgg caagaaccag atcgacaggt actacgaaac ttgtatcgga aaggataagg
     3541 gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat catcaccgga atgaactacg
     3601 accaattcga caagaaaagg agcgtcattg aggacaccgg cagggaaaac gccgagaggg
     3661 agaagtttaa aaagatcatc agcctgtacc tcaccgtgat ctaccacatc ctcaagaata
     3721 ttgtcaatat caacgccagg tacgtcatcg gattccattg cgtcgagcgt gatgctcaac
     3781 tgtacaagga gaaaggctac gacatcaatc tcaagaaact ggaagagaag ggattcagct
     3841 ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc cgataagaga aaggacgtgg
     3901 aaaaggagat ggctgaaaga gccaaggaga gcattgacag cctcgagagc gccaacccca
     3961 agctgtatgc caattacatc aaatacagcg acgagaagaa agccgaggag ttcaccaggc
     4021 agattaacag ggagaaggcc aaaaccgccc tgaacgccta cctgaggaac accaagtgga
     4081 atgtgatcat cagggaggac ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa
     4141 acaaggccgt ccacctggaa gtggccaggt atgtccacgc ctatatcaac gacattgccg
     4201 aggtcaattc ctacttccaa ctgtaccatt acatcatgca gagaattatc atgaatgaga
     4261 ggtacgagaa aagcagcgga aaggtgtccg agtacttcga cgctgtgaat gacgagaaga
     4321 agtacaacga taggctcctg aaactgctgt gtgtgccttt cggctactgt atccccaggt
     4381 ttaagaacct gagcatcgag gccctgttcg ataggaacga ggccgccaag ttcgacaagg
     4441 agaaaaagaa ggtgtccggc aattccggat ccggacctaa gaaaaagagg aaggtggcgg
     4501 ccgcttaccc atacgatgtt ccagattacg cttaagtcga caacttgttt attgcagctt
     4561 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4621 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgga
     4681 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4741 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4801 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4861 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4921 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4981 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5041 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5101 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     5161 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5221 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     5281 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5341 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     5401 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5521 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5581 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5641 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5701 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5761 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5821 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5881 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5941 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     6001 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     6061 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6121 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6181 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6241 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6301 ggcaacaacg ttgcgc
//
