LOCUS       sequence-331085-        6691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for expressing CasRx and control presgRNA for 
            RNA-editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6691)
  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 6691)
  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: pM123: pAAV-EFS-CasRx-control presgRNA
FEATURES             Location/Qualifiers
     source          1..6691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   70..199
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        214..462
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     214..234
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     385..404
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        464..499
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        530..565
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        638..849
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             869..889
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             899..3796
                     /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             3806..3826
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3836..3862
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3872..4006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3909..3928)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3963..3982
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4023..4163
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4023..4152
                     /label=AAV2 ITR
     rep_origin      4238..4693
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4325..4344)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4535..4556
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4710..4729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4829..4851
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4889..4907)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4975..5079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5080..5148,5149..5940)
                     /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(5298..5317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6111..6691,1..8)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6600..6619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
       61 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
      121 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
      181 tccatcacta ggggttcctg cggccgcacg cgtgagggcc tatttcccat gattccttca
      241 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
      301 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      361 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      421 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcaagtaa acccctacca
      481 actggtcggg gtttgaaact caccagaagc gtaccatact cacgaacagc aagtaaaccc
      541 ctaccaactg gtcggggttt gaaacttttt ttgaattctg atgcggtgaa gctttaggtc
      601 ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      661 gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag aaggtggcgc
      721 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      781 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
      841 agaacacagg tctagagcca ccatgagccc caagaagaag agaaaggtgg aggccagcat
      901 cgaaaaaaaa aagtccttcg ccaagggcat gggcgtgaag tccacactcg tgtccggctc
      961 caaagtgtac atgacaacct tcgccgaagg cagcgacgcc aggctggaaa agatcgtgga
     1021 gggcgacagc atcaggagcg tgaatgaggg cgaggccttc agcgctgaaa tggccgataa
     1081 aaacgccggc tataagatcg gcaacgccaa attcagccat cctaagggct acgccgtggt
     1141 ggctaacaac cctctgtata caggacccgt ccagcaggat atgctcggcc tgaaggaaac
     1201 tctggaaaag aggtacttcg gcgagagcgc tgatggcaat gacaatattt gtatccaggt
     1261 gatccataac atcctggaca ttgaaaaaat cctcgccgaa tacattacca acgccgccta
     1321 cgccgtcaac aatatctccg gcctggataa ggacattatt ggattcggca agttctccac
     1381 agtgtatacc tacgacgaat tcaaagaccc cgagcaccat agggccgctt tcaacaataa
     1441 cgataagctc atcaacgcca tcaaggccca gtatgacgag ttcgacaact tcctcgataa
     1501 ccccagactc ggctatttcg gccaggcctt tttcagcaag gagggcagaa attacatcat
     1561 caattacggc aacgaatgct atgacattct ggccctcctg agcggactga ggcactgggt
     1621 ggtccataac aacgaagaag agtccaggat ctccaggacc tggctctaca acctcgataa
     1681 gaacctcgac aacgaataca tctccaccct caactacctc tacgacagga tcaccaatga
     1741 gctgaccaac tccttctcca agaactccgc cgccaacgtg aactatattg ccgaaactct
     1801 gggaatcaac cctgccgaat tcgccgaaca atatttcaga ttcagcatta tgaaagagca
     1861 gaaaaacctc ggattcaata tcaccaagct cagggaagtg atgctggaca ggaaggatat
     1921 gtccgagatc aggaaaaatc ataaggtgtt cgactccatc aggaccaagg tctacaccat
     1981 gatggacttt gtgatttata ggtattacat cgaagaggat gccaaggtgg ctgccgccaa
     2041 taagtccctc cccgataatg agaagtccct gagcgagaag gatatctttg tgattaacct
     2101 gaggggctcc ttcaacgacg accagaagga tgccctctac tacgatgaag ctaatagaat
     2161 ttggagaaag ctcgaaaata tcatgcacaa catcaaggaa tttaggggaa acaagacaag
     2221 agagtataag aagaaggacg cccctagact gcccagaatc ctgcccgctg gccgtgatgt
     2281 ttccgccttc agcaaactca tgtatgccct gaccatgttc ctggatggca aggagatcaa
     2341 cgacctcctg accaccctga ttaataaatt cgataacatc cagagcttcc tgaaggtgat
     2401 gcctctcatc ggagtcaacg ctaagttcgt ggaggaatac gcctttttca aagactccgc
     2461 caagatcgcc gatgagctga ggctgatcaa gtccttcgct agaatgggag aacctattgc
     2521 cgatgccagg agggccatgt atatcgacgc catccgtatt ttaggaacca acctgtccta
     2581 tgatgagctc aaggccctcg ccgacacctt ttccctggac gagaacggaa acaagctcaa
     2641 gaaaggcaag cacggcatga gaaatttcat tattaataac gtgatcagca ataaaaggtt
     2701 ccactacctg atcagatacg gtgatcctgc ccacctccat gagatcgcca aaaacgaggc
     2761 cgtggtgaag ttcgtgctcg gcaggatcgc tgacatccag aaaaaacagg gccagaacgg
     2821 caagaaccag atcgacaggt actacgaaac ttgtatcgga aaggataagg gcaagagcgt
     2881 gagcgaaaag gtggacgctc tcacaaagat catcaccgga atgaactacg accaattcga
     2941 caagaaaagg agcgtcattg aggacaccgg cagggaaaac gccgagaggg agaagtttaa
     3001 aaagatcatc agcctgtacc tcaccgtgat ctaccacatc ctcaagaata ttgtcaatat
     3061 caacgccagg tacgtcatcg gattccattg cgtcgagcgt gatgctcaac tgtacaagga
     3121 gaaaggctac gacatcaatc tcaagaaact ggaagagaag ggattcagct ccgtcaccaa
     3181 gctctgcgct ggcattgatg aaactgcccc cgataagaga aaggacgtgg aaaaggagat
     3241 ggctgaaaga gccaaggaga gcattgacag cctcgagagc gccaacccca agctgtatgc
     3301 caattacatc aaatacagcg acgagaagaa agccgaggag ttcaccaggc agattaacag
     3361 ggagaaggcc aaaaccgccc tgaacgccta cctgaggaac accaagtgga atgtgatcat
     3421 cagggaggac ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa acaaggccgt
     3481 ccacctggaa gtggccaggt atgtccacgc ctatatcaac gacattgccg aggtcaattc
     3541 ctacttccaa ctgtaccatt acatcatgca gagaattatc atgaatgaga ggtacgagaa
     3601 aagcagcgga aaggtgtccg agtacttcga cgctgtgaat gacgagaaga agtacaacga
     3661 taggctcctg aaactgctgt gtgtgccttt cggctactgt atccccaggt ttaagaacct
     3721 gagcatcgag gccctgttcg ataggaacga ggccgccaag ttcgacaagg agaaaaagaa
     3781 ggtgtccggc aattccggat ccggacctaa gaaaaagagg aaggtggcgg ccgcttaccc
     3841 atacgatgtt ccagattacg cttaagtcga caacttgttt attgcagctt ataatggtta
     3901 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3961 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgga ccgagcggcc
     4021 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4081 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4141 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4201 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4261 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4321 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4381 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4441 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4501 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4561 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     4621 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4681 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4741 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4801 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4861 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4921 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4981 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5041 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5101 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5161 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5221 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5281 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5341 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5401 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5461 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5521 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5581 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5641 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5701 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5761 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5821 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5881 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5941 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6001 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6061 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6121 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6181 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6241 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6301 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6361 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6421 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6481 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6541 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6601 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6661 tttttgtgat gctcgtcagg ggggcggagc c
//
