LOCUS       sequence-401407-        7331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active RfxCas13d with an array of three VEGFA 
            mRNA targeting gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7331)
  AUTHORS   Kumar S, Hsiao YW, Wong VHY, Aubin D, Wang JH, Lisowski L, Rakoczy 
            EP, Li F, Alarcon-Martinez L, Gonzalez-Cordero A, Bui BV, Liu GS
  TITLE     Characterization of RNA editing and gene therapy with a compact 
            CRISPR-Cas13 in the retina.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Nov 5;121(45):e2408345121. doi: 
            10.1073/pnas.2408345121. Epub 2024 Oct 30.
  PUBMED    39475642
REFERENCE   2  (bases 1 to 7331)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-11-5"; volume: "121(45):e2408345121. 
            doi"; pages: " 10.1073/pnas.2408345121. Epub 2024 Oct 30"
COMMENT     Sequence Label: pC131: pAAV.CMV-CasRx-VEGFA sgRNA array
FEATURES             Location/Qualifiers
     source          1..7331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        169..417
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     169..189
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     340..359
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..454
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        485..520
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        551..586
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        617..652
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     protein_bind    681..705
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        766..1069
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1070..1273
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1224..1244
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1295..1319)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1331..1351
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1361..4258
                     /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             4268..4288
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4298..4324
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(4328..4348)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(4373..4390)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4379..4586
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4594..4734
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4594..4723
                     /label=AAV2 ITR
     rep_origin      4809..5264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4896..4915)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5106..5127
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5281..5300)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5400..5422
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5460..5478)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5546..5650
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5651..5719,5720..6511)
                     /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(5869..5888)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6682..7270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7171..7190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgga gggcctattt
      181 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
      241 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      301 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      361 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca
      421 agtaaacccc taccaactgg tcggggtttg aaactatgtg ctggccttgg tgaggtttga
      481 tccgcaagta aacccctacc aactggtcgg ggtttgaaac cacttcgtga tgattctgcc
      541 ctcctccttc caagtaaacc cctaccaact ggtcggggtt tgaaactctg cattcacatt
      601 tgttgtgctg taggaacaag taaaccccta ccaactggtc ggggtttgaa actttttttg
      661 aattctgatg cggtgaagct tcaagtttgt acaaaaaagc aggcttagtt attaatagta
      721 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      781 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      841 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      901 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      961 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     1021 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     1081 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1141 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1201 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1261 tataagcaga gctggtttag tgaaccgtca gatcacccag ctttcttgta caaagtgggc
     1321 caccatgagc cccaagaaga agagaaaggt ggaggccagc atcgaaaaaa aaaagtcctt
     1381 cgccaagggc atgggcgtga agtccacact cgtgtccggc tccaaagtgt acatgacaac
     1441 cttcgccgaa ggcagcgacg ccaggctgga aaagatcgtg gagggcgaca gcatcaggag
     1501 cgtgaatgag ggcgaggcct tcagcgctga aatggccgat aaaaacgccg gctataagat
     1561 cggcaacgcc aaattcagcc atcctaaggg ctacgccgtg gtggctaaca accctctgta
     1621 tacaggaccc gtccagcagg atatgctcgg cctgaaggaa actctggaaa agaggtactt
     1681 cggcgagagc gctgatggca atgacaatat ttgtatccag gtgatccata acatcctgga
     1741 cattgaaaaa atcctcgccg aatacattac caacgccgcc tacgccgtca acaatatctc
     1801 cggcctggat aaggacatta ttggattcgg caagttctcc acagtgtata cctacgacga
     1861 attcaaagac cccgagcacc atagggccgc tttcaacaat aacgataagc tcatcaacgc
     1921 catcaaggcc cagtatgacg agttcgacaa cttcctcgat aaccccagac tcggctattt
     1981 cggccaggcc tttttcagca aggagggcag aaattacatc atcaattacg gcaacgaatg
     2041 ctatgacatt ctggccctcc tgagcggact gaggcactgg gtggtccata acaacgaaga
     2101 agagtccagg atctccagga cctggctcta caacctcgat aagaacctcg acaacgaata
     2161 catctccacc ctcaactacc tctacgacag gatcaccaat gagctgacca actccttctc
     2221 caagaactcc gccgccaacg tgaactatat tgccgaaact ctgggaatca accctgccga
     2281 attcgccgaa caatatttca gattcagcat tatgaaagag cagaaaaacc tcggattcaa
     2341 tatcaccaag ctcagggaag tgatgctgga caggaaggat atgtccgaga tcaggaaaaa
     2401 tcataaggtg ttcgactcca tcaggaccaa ggtctacacc atgatggact ttgtgattta
     2461 taggtattac atcgaagagg atgccaaggt ggctgccgcc aataagtccc tccccgataa
     2521 tgagaagtcc ctgagcgaga aggatatctt tgtgattaac ctgaggggct ccttcaacga
     2581 cgaccagaag gatgccctct actacgatga agctaataga atttggagaa agctcgaaaa
     2641 tatcatgcac aacatcaagg aatttagggg aaacaagaca agagagtata agaagaagga
     2701 cgcccctaga ctgcccagaa tcctgcccgc tggccgtgat gtttccgcct tcagcaaact
     2761 catgtatgcc ctgaccatgt tcctggatgg caaggagatc aacgacctcc tgaccaccct
     2821 gattaataaa ttcgataaca tccagagctt cctgaaggtg atgcctctca tcggagtcaa
     2881 cgctaagttc gtggaggaat acgccttttt caaagactcc gccaagatcg ccgatgagct
     2941 gaggctgatc aagtccttcg ctagaatggg agaacctatt gccgatgcca ggagggccat
     3001 gtatatcgac gccatccgta ttttaggaac caacctgtcc tatgatgagc tcaaggccct
     3061 cgccgacacc ttttccctgg acgagaacgg aaacaagctc aagaaaggca agcacggcat
     3121 gagaaatttc attattaata acgtgatcag caataaaagg ttccactacc tgatcagata
     3181 cggtgatcct gcccacctcc atgagatcgc caaaaacgag gccgtggtga agttcgtgct
     3241 cggcaggatc gctgacatcc agaaaaaaca gggccagaac ggcaagaacc agatcgacag
     3301 gtactacgaa acttgtatcg gaaaggataa gggcaagagc gtgagcgaaa aggtggacgc
     3361 tctcacaaag atcatcaccg gaatgaacta cgaccaattc gacaagaaaa ggagcgtcat
     3421 tgaggacacc ggcagggaaa acgccgagag ggagaagttt aaaaagatca tcagcctgta
     3481 cctcaccgtg atctaccaca tcctcaagaa tattgtcaat atcaacgcca ggtacgtcat
     3541 cggattccat tgcgtcgagc gtgatgctca actgtacaag gagaaaggct acgacatcaa
     3601 tctcaagaaa ctggaagaga agggattcag ctccgtcacc aagctctgcg ctggcattga
     3661 tgaaactgcc cccgataaga gaaaggacgt ggaaaaggag atggctgaaa gagccaagga
     3721 gagcattgac agcctcgaga gcgccaaccc caagctgtat gccaattaca tcaaatacag
     3781 cgacgagaag aaagccgagg agttcaccag gcagattaac agggagaagg ccaaaaccgc
     3841 cctgaacgcc tacctgagga acaccaagtg gaatgtgatc atcagggagg acctcctgag
     3901 aattgacaac aagacatgta ccctgttcag aaacaaggcc gtccacctgg aagtggccag
     3961 gtatgtccac gcctatatca acgacattgc cgaggtcaat tcctacttcc aactgtacca
     4021 ttacatcatg cagagaatta tcatgaatga gaggtacgag aaaagcagcg gaaaggtgtc
     4081 cgagtacttc gacgctgtga atgacgagaa gaagtacaac gataggctcc tgaaactgct
     4141 gtgtgtgcct ttcggctact gtatccccag gtttaagaac ctgagcatcg aggccctgtt
     4201 cgataggaac gaggccgcca agttcgacaa ggagaaaaag aaggtgtccg gcaattccgg
     4261 atccggacct aagaaaaaga ggaaggtggc ggccgcttac ccatacgatg ttccagatta
     4321 cgcttaacaa ctttattata catagttgga attcctagag ctcgctgatc agcctcgact
     4381 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4441 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4501 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4561 gaagagaata gcaggcatgc tggggagggc cgcaggaacc cctagtgatg gagttggcca
     4621 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4681 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     4741 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4801 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4861 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     4921 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4981 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     5041 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5101 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     5161 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     5221 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     5281 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     5341 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5401 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     5461 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5521 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5581 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5641 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5701 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5761 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5821 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5881 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5941 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6001 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6061 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6121 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6181 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6241 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6301 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6361 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6421 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6481 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6541 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6601 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6661 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6721 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6781 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6841 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6901 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6961 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     7021 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     7081 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7141 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7201 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7261 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7321 tgctcacatg t
//
