LOCUS       sequence-331086-        6691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for expressing CasRx and human VEGFA targeting 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: pM124: pAAV-EFS-CasRx-VEGFA presgRNA
FEATURES             Location/Qualifiers
     source          1..6691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..73
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   215..344
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        359..607
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     359..379
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     530..549
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        609..644
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        675..710
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        783..994
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1014..1034
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1044..3941
                     /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             3951..3971
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3981..4007
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4017..4151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4054..4073)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4108..4127
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4168..4308
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4168..4297
                     /label=AAV2 ITR
     rep_origin      4383..4838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4470..4489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4680..4701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4855..4874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4974..4996
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5034..5052)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5120..5224
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5225..5293,5294..6085)
                     /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(5443..5462)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6256..6691,1..153)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
       61 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      121 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      181 ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg
      241 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
      301 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtga
      361 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      421 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      481 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      541 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      601 aaacaccgca agtaaacccc taccaactgg tcggggtttg aaactatgtg ctggccttgg
      661 tgaggtttga tccgcaagta aacccctacc aactggtcgg ggtttgaaac tttttttgaa
      721 ttctgatgcg gtgaagcttt aggtcttgaa aggagtggga attggctccg gtgcccgtca
      781 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
      841 atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
      901 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
      961 tctttttcgc aacgggtttg ccgccagaac acaggtctag agccaccatg agccccaaga
     1021 agaagagaaa ggtggaggcc agcatcgaaa aaaaaaagtc cttcgccaag ggcatgggcg
     1081 tgaagtccac actcgtgtcc ggctccaaag tgtacatgac aaccttcgcc gaaggcagcg
     1141 acgccaggct ggaaaagatc gtggagggcg acagcatcag gagcgtgaat gagggcgagg
     1201 ccttcagcgc tgaaatggcc gataaaaacg ccggctataa gatcggcaac gccaaattca
     1261 gccatcctaa gggctacgcc gtggtggcta acaaccctct gtatacagga cccgtccagc
     1321 aggatatgct cggcctgaag gaaactctgg aaaagaggta cttcggcgag agcgctgatg
     1381 gcaatgacaa tatttgtatc caggtgatcc ataacatcct ggacattgaa aaaatcctcg
     1441 ccgaatacat taccaacgcc gcctacgccg tcaacaatat ctccggcctg gataaggaca
     1501 ttattggatt cggcaagttc tccacagtgt atacctacga cgaattcaaa gaccccgagc
     1561 accatagggc cgctttcaac aataacgata agctcatcaa cgccatcaag gcccagtatg
     1621 acgagttcga caacttcctc gataacccca gactcggcta tttcggccag gcctttttca
     1681 gcaaggaggg cagaaattac atcatcaatt acggcaacga atgctatgac attctggccc
     1741 tcctgagcgg actgaggcac tgggtggtcc ataacaacga agaagagtcc aggatctcca
     1801 ggacctggct ctacaacctc gataagaacc tcgacaacga atacatctcc accctcaact
     1861 acctctacga caggatcacc aatgagctga ccaactcctt ctccaagaac tccgccgcca
     1921 acgtgaacta tattgccgaa actctgggaa tcaaccctgc cgaattcgcc gaacaatatt
     1981 tcagattcag cattatgaaa gagcagaaaa acctcggatt caatatcacc aagctcaggg
     2041 aagtgatgct ggacaggaag gatatgtccg agatcaggaa aaatcataag gtgttcgact
     2101 ccatcaggac caaggtctac accatgatgg actttgtgat ttataggtat tacatcgaag
     2161 aggatgccaa ggtggctgcc gccaataagt ccctccccga taatgagaag tccctgagcg
     2221 agaaggatat ctttgtgatt aacctgaggg gctccttcaa cgacgaccag aaggatgccc
     2281 tctactacga tgaagctaat agaatttgga gaaagctcga aaatatcatg cacaacatca
     2341 aggaatttag gggaaacaag acaagagagt ataagaagaa ggacgcccct agactgccca
     2401 gaatcctgcc cgctggccgt gatgtttccg ccttcagcaa actcatgtat gccctgacca
     2461 tgttcctgga tggcaaggag atcaacgacc tcctgaccac cctgattaat aaattcgata
     2521 acatccagag cttcctgaag gtgatgcctc tcatcggagt caacgctaag ttcgtggagg
     2581 aatacgcctt tttcaaagac tccgccaaga tcgccgatga gctgaggctg atcaagtcct
     2641 tcgctagaat gggagaacct attgccgatg ccaggagggc catgtatatc gacgccatcc
     2701 gtattttagg aaccaacctg tcctatgatg agctcaaggc cctcgccgac accttttccc
     2761 tggacgagaa cggaaacaag ctcaagaaag gcaagcacgg catgagaaat ttcattatta
     2821 ataacgtgat cagcaataaa aggttccact acctgatcag atacggtgat cctgcccacc
     2881 tccatgagat cgccaaaaac gaggccgtgg tgaagttcgt gctcggcagg atcgctgaca
     2941 tccagaaaaa acagggccag aacggcaaga accagatcga caggtactac gaaacttgta
     3001 tcggaaagga taagggcaag agcgtgagcg aaaaggtgga cgctctcaca aagatcatca
     3061 ccggaatgaa ctacgaccaa ttcgacaaga aaaggagcgt cattgaggac accggcaggg
     3121 aaaacgccga gagggagaag tttaaaaaga tcatcagcct gtacctcacc gtgatctacc
     3181 acatcctcaa gaatattgtc aatatcaacg ccaggtacgt catcggattc cattgcgtcg
     3241 agcgtgatgc tcaactgtac aaggagaaag gctacgacat caatctcaag aaactggaag
     3301 agaagggatt cagctccgtc accaagctct gcgctggcat tgatgaaact gcccccgata
     3361 agagaaagga cgtggaaaag gagatggctg aaagagccaa ggagagcatt gacagcctcg
     3421 agagcgccaa ccccaagctg tatgccaatt acatcaaata cagcgacgag aagaaagccg
     3481 aggagttcac caggcagatt aacagggaga aggccaaaac cgccctgaac gcctacctga
     3541 ggaacaccaa gtggaatgtg atcatcaggg aggacctcct gagaattgac aacaagacat
     3601 gtaccctgtt cagaaacaag gccgtccacc tggaagtggc caggtatgtc cacgcctata
     3661 tcaacgacat tgccgaggtc aattcctact tccaactgta ccattacatc atgcagagaa
     3721 ttatcatgaa tgagaggtac gagaaaagca gcggaaaggt gtccgagtac ttcgacgctg
     3781 tgaatgacga gaagaagtac aacgataggc tcctgaaact gctgtgtgtg cctttcggct
     3841 actgtatccc caggtttaag aacctgagca tcgaggccct gttcgatagg aacgaggccg
     3901 ccaagttcga caaggagaaa aagaaggtgt ccggcaattc cggatccgga cctaagaaaa
     3961 agaggaaggt ggcggccgct tacccatacg atgttccaga ttacgcttaa gtcgacaact
     4021 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4081 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4141 atgtctggat ccggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     4201 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     4261 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     4321 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4381 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4441 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4501 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4561 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     4621 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4681 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     4741 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     4801 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     4861 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     4921 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4981 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     5041 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5101 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5161 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5221 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5281 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5341 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5401 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5461 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5521 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5581 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5641 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5701 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5761 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5821 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5881 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5941 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6001 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6061 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6121 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6181 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6241 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6301 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6361 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6421 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6481 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6541 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6601 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6661 cacgcttccc gaagggagaa aggcggacag g
//
