LOCUS       sequence-455884-        6578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active Cas13e with VEGFA mRNA targeting gRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6578)
  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 6578)
  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: pC369: pAAV.CMV-Cas13e-VEGFA sgRNA3
FEATURES             Location/Qualifiers
     source          1..6578
                     /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"
     enhancer        610..913
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        914..1117
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1068..1088
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1138..1162
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1172..1192
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3515..3535
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3542..3568
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3572..3596)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3626..3833
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3841..3981
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3841..3970
                     /label=AAV2 ITR
     rep_origin      4056..4511
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4143..4162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4353..4374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4528..4547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4647..4669
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4707..4725)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4793..4897
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4898..4966,4967..5758)
                     /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(5116..5135)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5929..6517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6418..6437
                     /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 aaacaccggc
      421 tggagcagcc cccgatttgt ggggtgatta cagctctgca ttcacatttg ttgtgctgta
      481 ggaagctgga gcagcccccg atttgtgggg tgattacagc tttttttgaa ttctgatgcg
      541 gtgaagcttt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      601 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      661 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      721 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      781 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      841 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      901 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      961 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     1021 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1081 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcca
     1141 agtttgtaca aaaaagcagg ctgccaccat gccaaagaag aaacggaagg tcgctcaggt
     1201 ctctaagcag acatccaaga agagggaact ttccattgat gaatatcaag gtgccagaaa
     1261 atggtgtttt acaatcgcct ttaataaggc cttggtcaat cgggacaaga atgacggact
     1321 tttcgttgaa tcccttctcc gccacgaaaa atactccaag catgattggt acgatgagga
     1381 caccagggca ttgatcaaat gtagcacaca agccgcaaat gctaaggcag aggctcttcg
     1441 aaattacttc tcacattaca ggcacagtcc agggtgcttg accttcaccg cagaggacga
     1501 gttgcggact attatggagc gggcatacga aagggccata ttcgaatgta gacgacgcga
     1561 aacagaagtc attattgagt tcccctcact gttcgagggc gataggataa ctacagctgg
     1621 ggtagtcttt tttgtctctt tcttcgttga aagacgagtg ctcgaccgac tctacggagc
     1681 cgtgagcggt ctgaaaaaga atgagggcca atacaaactc acccgaaagg cactctccat
     1741 gtactgtctt aaggattccc gatttaccaa ggcatgggac aaaagggtat tgttgttcag
     1801 ggatatcctg gcacagttgg ggcgaattcc cgccgaagca tatgaatact accacggcga
     1861 acagggtgat aaaaaacgcg ctaatgataa cgagggcaca aatccaaagc gccataaaga
     1921 caaattcatt gaattcgcat tgcattatct tgaagcccag cacagcgaaa tctgcttcgg
     1981 taggcgccac attgtacgag aagaggctgg tgctggagat gaacataaga agcaccgcac
     2041 caaggggaag gtagttgtcg attttagtaa gaaagatgaa gaccagtcat attacatatc
     2101 caagaataac gtaattgtgc gcattgataa gaacgccgga ccacgctctt atagaatggg
     2161 gctgaatgag ctcaagtact tggtcttgct tagcctccag ggaaaagggg atgacgctat
     2221 tgctaagctc tatcgctata gacaacacgt tgagaatatc ctcgacgttg tgaaagttac
     2281 agataaggat aatcatgtat tcctgcctag atttgtgttg gaacagcacg gcattggtag
     2341 gaaggcattc aagcaaagga tcgatggaag agtcaagcat gtgcgcggag tgtgggaaaa
     2401 aaaaaaagca gccactaacg agatgaccct gcacgaaaaa gctagggata tccttcagta
     2461 tgtaaacgaa aactgtactc gctcctttaa tcctggagaa tacaatagac tgcttgtgtg
     2521 ccttgttggg aaggatgtag agaactttca agccggcctt aagcgactcc agttggctga
     2581 acggatagat ggacgagtgt atagtatatt cgcacagact tctacaatca atgaaatgca
     2641 tcaagtcgtt tgtgaccaaa ttttgaacag gctttgcagg atcggtgatc aaaagctcta
     2701 tgactatgtc gggctgggta agaaggatga aatagattac aaacagaagg tcgcctggtt
     2761 taaggagcac atttccatca gacgggggtt tttgcggaag aaattttggt acgattctaa
     2821 aaaaggattt gcaaagctgg tggaagaaca tttggagagc ggaggcgggc agcgcgacgt
     2881 aggtcttgat aagaaatact accacataga cgcaattggg cggttcgagg gagcaaaccc
     2941 tgccttgtat gaaaccctcg cccgagatag attgtgcttg atgatggcac aatattttct
     3001 gggttcagta cggaaggaac ttggtaacaa gattgtttgg agcaatgaca gcatcgaatt
     3061 gcctgttgaa gggagtgtcg gtaatgaaaa gtccattgta ttttccgtga gtgactatgg
     3121 caagttgtat gttcttgacg atgcagaatt ccttgggcga atctgcgagt acttcatgcc
     3181 ccacgaaaag ggtaaaatac gctatcatac agtttatgag aaaggctttc gagcatataa
     3241 tgacctgcaa aagaagtgtg tcgaagcagt ccttgctttc gaagaaaagg tcgtaaaagc
     3301 aaagaagatg tccgaaaaag agggcgccca ttacattgac ttcagagaga tccttgccca
     3361 gacaatgtgt aaggaagccg agaaaactgc tgtgaataag gtaaggagag cctttttcca
     3421 tcaccacctc aagttcgtca tagacgaatt tggattgttt tctgatgtga tgaaaaaata
     3481 tgggattgag aaagaatgga agtttccagt gaagcccaag aaaaaaagaa aagtatcctc
     3541 ctacccatac gatgttccag attacgcttg aacccagctt tcttgtacaa agtgggaatt
     3601 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     3661 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     3721 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3781 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagggccgc
     3841 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3901 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     3961 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     4021 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     4081 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     4141 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4201 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4261 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4321 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4381 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     4441 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4501 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4561 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4621 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4681 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4741 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4801 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4861 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4921 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4981 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5041 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5101 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5161 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5221 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5281 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5341 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5401 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5461 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5521 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5581 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     5641 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5701 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5761 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5821 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5881 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5941 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6001 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6061 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6121 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6181 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6241 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6301 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6361 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6421 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6481 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6541 acggttcctg gccttttgct ggccttttgc tcacatgt
//
