LOCUS       sequence-331090-        6823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for expressing CasRx and human VEGFA targeting presgRNA 
            array for RNA-editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6823)
  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 6823)
  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: pM125: pAAV-EFS-CasRx-VEGFA array presgRNA
FEATURES             Location/Qualifiers
     source          1..6823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   26..155
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        170..418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     170..190
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     341..360
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        420..455
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        486..521
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        552..587
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        618..653
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     promoter        726..937
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             957..977
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             987..3884
                     /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             3894..3914
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3924..3950
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3960..4094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3997..4016)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4051..4070
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4111..4251
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4111..4240
                     /label=AAV2 ITR
     rep_origin      4326..4781
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4413..4432)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4623..4644
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4798..4817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4917..4939
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4977..4995)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5063..5167
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5168..5236,5237..6028)
                     /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(5386..5405)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6199..6787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6688..6707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag
       61 gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
      121 agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtg agggcctatt
      181 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      241 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      301 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      361 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc
      421 aagtaaaccc ctaccaactg gtcggggttt gaaactatgt gctggccttg gtgaggtttg
      481 atccgcaagt aaacccctac caactggtcg gggtttgaaa ccacttcgtg atgattctgc
      541 cctcctcctt ccaagtaaac ccctaccaac tggtcggggt ttgaaactct gcattcacat
      601 ttgttgtgct gtaggaacaa gtaaacccct accaactggt cggggtttga aacttttttt
      661 gaattctgat gcggtgaagc tttaggtctt gaaaggagtg ggaattggct ccggtgcccg
      721 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      781 ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      841 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      901 cgttcttttt cgcaacgggt ttgccgccag aacacaggtc tagagccacc atgagcccca
      961 agaagaagag aaaggtggag gccagcatcg aaaaaaaaaa gtccttcgcc aagggcatgg
     1021 gcgtgaagtc cacactcgtg tccggctcca aagtgtacat gacaaccttc gccgaaggca
     1081 gcgacgccag gctggaaaag atcgtggagg gcgacagcat caggagcgtg aatgagggcg
     1141 aggccttcag cgctgaaatg gccgataaaa acgccggcta taagatcggc aacgccaaat
     1201 tcagccatcc taagggctac gccgtggtgg ctaacaaccc tctgtataca ggacccgtcc
     1261 agcaggatat gctcggcctg aaggaaactc tggaaaagag gtacttcggc gagagcgctg
     1321 atggcaatga caatatttgt atccaggtga tccataacat cctggacatt gaaaaaatcc
     1381 tcgccgaata cattaccaac gccgcctacg ccgtcaacaa tatctccggc ctggataagg
     1441 acattattgg attcggcaag ttctccacag tgtataccta cgacgaattc aaagaccccg
     1501 agcaccatag ggccgctttc aacaataacg ataagctcat caacgccatc aaggcccagt
     1561 atgacgagtt cgacaacttc ctcgataacc ccagactcgg ctatttcggc caggcctttt
     1621 tcagcaagga gggcagaaat tacatcatca attacggcaa cgaatgctat gacattctgg
     1681 ccctcctgag cggactgagg cactgggtgg tccataacaa cgaagaagag tccaggatct
     1741 ccaggacctg gctctacaac ctcgataaga acctcgacaa cgaatacatc tccaccctca
     1801 actacctcta cgacaggatc accaatgagc tgaccaactc cttctccaag aactccgccg
     1861 ccaacgtgaa ctatattgcc gaaactctgg gaatcaaccc tgccgaattc gccgaacaat
     1921 atttcagatt cagcattatg aaagagcaga aaaacctcgg attcaatatc accaagctca
     1981 gggaagtgat gctggacagg aaggatatgt ccgagatcag gaaaaatcat aaggtgttcg
     2041 actccatcag gaccaaggtc tacaccatga tggactttgt gatttatagg tattacatcg
     2101 aagaggatgc caaggtggct gccgccaata agtccctccc cgataatgag aagtccctga
     2161 gcgagaagga tatctttgtg attaacctga ggggctcctt caacgacgac cagaaggatg
     2221 ccctctacta cgatgaagct aatagaattt ggagaaagct cgaaaatatc atgcacaaca
     2281 tcaaggaatt taggggaaac aagacaagag agtataagaa gaaggacgcc cctagactgc
     2341 ccagaatcct gcccgctggc cgtgatgttt ccgccttcag caaactcatg tatgccctga
     2401 ccatgttcct ggatggcaag gagatcaacg acctcctgac caccctgatt aataaattcg
     2461 ataacatcca gagcttcctg aaggtgatgc ctctcatcgg agtcaacgct aagttcgtgg
     2521 aggaatacgc ctttttcaaa gactccgcca agatcgccga tgagctgagg ctgatcaagt
     2581 ccttcgctag aatgggagaa cctattgccg atgccaggag ggccatgtat atcgacgcca
     2641 tccgtatttt aggaaccaac ctgtcctatg atgagctcaa ggccctcgcc gacacctttt
     2701 ccctggacga gaacggaaac aagctcaaga aaggcaagca cggcatgaga aatttcatta
     2761 ttaataacgt gatcagcaat aaaaggttcc actacctgat cagatacggt gatcctgccc
     2821 acctccatga gatcgccaaa aacgaggccg tggtgaagtt cgtgctcggc aggatcgctg
     2881 acatccagaa aaaacagggc cagaacggca agaaccagat cgacaggtac tacgaaactt
     2941 gtatcggaaa ggataagggc aagagcgtga gcgaaaaggt ggacgctctc acaaagatca
     3001 tcaccggaat gaactacgac caattcgaca agaaaaggag cgtcattgag gacaccggca
     3061 gggaaaacgc cgagagggag aagtttaaaa agatcatcag cctgtacctc accgtgatct
     3121 accacatcct caagaatatt gtcaatatca acgccaggta cgtcatcgga ttccattgcg
     3181 tcgagcgtga tgctcaactg tacaaggaga aaggctacga catcaatctc aagaaactgg
     3241 aagagaaggg attcagctcc gtcaccaagc tctgcgctgg cattgatgaa actgcccccg
     3301 ataagagaaa ggacgtggaa aaggagatgg ctgaaagagc caaggagagc attgacagcc
     3361 tcgagagcgc caaccccaag ctgtatgcca attacatcaa atacagcgac gagaagaaag
     3421 ccgaggagtt caccaggcag attaacaggg agaaggccaa aaccgccctg aacgcctacc
     3481 tgaggaacac caagtggaat gtgatcatca gggaggacct cctgagaatt gacaacaaga
     3541 catgtaccct gttcagaaac aaggccgtcc acctggaagt ggccaggtat gtccacgcct
     3601 atatcaacga cattgccgag gtcaattcct acttccaact gtaccattac atcatgcaga
     3661 gaattatcat gaatgagagg tacgagaaaa gcagcggaaa ggtgtccgag tacttcgacg
     3721 ctgtgaatga cgagaagaag tacaacgata ggctcctgaa actgctgtgt gtgcctttcg
     3781 gctactgtat ccccaggttt aagaacctga gcatcgaggc cctgttcgat aggaacgagg
     3841 ccgccaagtt cgacaaggag aaaaagaagg tgtccggcaa ttccggatcc ggacctaaga
     3901 aaaagaggaa ggtggcggcc gcttacccat acgatgttcc agattacgct taagtcgaca
     3961 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4021 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4081 atcatgtctg gatccggacc gagcggccgc aggaacccct agtgatggag ttggccactc
     4141 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4201 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4261 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4321 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4381 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     4441 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4501 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4561 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4621 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     4681 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     4741 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     4801 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     4861 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4921 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     4981 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5041 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5101 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5161 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5221 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5281 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5341 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5401 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5461 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5521 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5581 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5641 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5701 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5761 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5821 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5881 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5941 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6001 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6061 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6121 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6181 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6241 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6301 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6361 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6421 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6481 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6541 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6601 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6661 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6721 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6781 atggaaaaac gccagcaacg cggccttttt acggttcctg gcc
//
