LOCUS       sequence-401475-        6791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active Cas13e 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 6791)
  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 6791)
  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: pM150: pAAV.CMV-Cas13e-VEGFA sgRNA array
FEATURES             Location/Qualifiers
     source          1..6791
                     /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"
     protein_bind    741..765
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        826..1129
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1130..1333
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1284..1304
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1355..1379)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1388..1408
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3731..3751
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3758..3784
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3788..3808)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3839..4046
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4054..4194
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4054..4183
                     /label=AAV2 ITR
     rep_origin      4269..4724
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4356..4375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4566..4587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4741..4760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4860..4882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4920..4938)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5006..5110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5111..5179,5180..5971)
                     /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(5329..5348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6142..6730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6631..6650
                     /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 cagctatgtg ctggccttgg tgaggtttga
      481 tccggctgga gcagcccccg atttgtgggg tgattacagc cacttcgtga tgattctgcc
      541 ctcctccttc gctggagcag cccccgattt gtggggtgat tacagctctg cattcacatt
      601 tgttgtgctg taggaagctg gagcagcccc cgatttgtgg ggtgattaca gctttttttg
      661 aattctgatg cggtgaagct ttagttatta atagtaatca attacggggt cattagttca
      721 tagcccatat atggagttcc gcaagtttgt acaaaaaagc aggcttagtt attaatagta
      781 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      841 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      901 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      961 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     1021 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     1081 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     1141 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1201 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1261 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1321 tataagcaga gctggtttag tgaaccgtca gatcacccag ctttcttgta caaagtgggc
     1381 caccatgcca aagaagaaac ggaaggtcgc tcaggtctct aagcagacat ccaagaagag
     1441 ggaactttcc attgatgaat atcaaggtgc cagaaaatgg tgttttacaa tcgcctttaa
     1501 taaggccttg gtcaatcggg acaagaatga cggacttttc gttgaatccc ttctccgcca
     1561 cgaaaaatac tccaagcatg attggtacga tgaggacacc agggcattga tcaaatgtag
     1621 cacacaagcc gcaaatgcta aggcagaggc tcttcgaaat tacttctcac attacaggca
     1681 cagtccaggg tgcttgacct tcaccgcaga ggacgagttg cggactatta tggagcgggc
     1741 atacgaaagg gccatattcg aatgtagacg acgcgaaaca gaagtcatta ttgagttccc
     1801 ctcactgttc gagggcgata ggataactac agctggggta gtcttttttg tctctttctt
     1861 cgttgaaaga cgagtgctcg accgactcta cggagccgtg agcggtctga aaaagaatga
     1921 gggccaatac aaactcaccc gaaaggcact ctccatgtac tgtcttaagg attcccgatt
     1981 taccaaggca tgggacaaaa gggtattgtt gttcagggat atcctggcac agttggggcg
     2041 aattcccgcc gaagcatatg aatactacca cggcgaacag ggtgataaaa aacgcgctaa
     2101 tgataacgag ggcacaaatc caaagcgcca taaagacaaa ttcattgaat tcgcattgca
     2161 ttatcttgaa gcccagcaca gcgaaatctg cttcggtagg cgccacattg tacgagaaga
     2221 ggctggtgct ggagatgaac ataagaagca ccgcaccaag gggaaggtag ttgtcgattt
     2281 tagtaagaaa gatgaagacc agtcatatta catatccaag aataacgtaa ttgtgcgcat
     2341 tgataagaac gccggaccac gctcttatag aatggggctg aatgagctca agtacttggt
     2401 cttgcttagc ctccagggaa aaggggatga cgctattgct aagctctatc gctatagaca
     2461 acacgttgag aatatcctcg acgttgtgaa agttacagat aaggataatc atgtattcct
     2521 gcctagattt gtgttggaac agcacggcat tggtaggaag gcattcaagc aaaggatcga
     2581 tggaagagtc aagcatgtgc gcggagtgtg ggaaaaaaaa aaagcagcca ctaacgagat
     2641 gaccctgcac gaaaaagcta gggatatcct tcagtatgta aacgaaaact gtactcgctc
     2701 ctttaatcct ggagaataca atagactgct tgtgtgcctt gttgggaagg atgtagagaa
     2761 ctttcaagcc ggccttaagc gactccagtt ggctgaacgg atagatggac gagtgtatag
     2821 tatattcgca cagacttcta caatcaatga aatgcatcaa gtcgtttgtg accaaatttt
     2881 gaacaggctt tgcaggatcg gtgatcaaaa gctctatgac tatgtcgggc tgggtaagaa
     2941 ggatgaaata gattacaaac agaaggtcgc ctggtttaag gagcacattt ccatcagacg
     3001 ggggtttttg cggaagaaat tttggtacga ttctaaaaaa ggatttgcaa agctggtgga
     3061 agaacatttg gagagcggag gcgggcagcg cgacgtaggt cttgataaga aatactacca
     3121 catagacgca attgggcggt tcgagggagc aaaccctgcc ttgtatgaaa ccctcgcccg
     3181 agatagattg tgcttgatga tggcacaata ttttctgggt tcagtacgga aggaacttgg
     3241 taacaagatt gtttggagca atgacagcat cgaattgcct gttgaaggga gtgtcggtaa
     3301 tgaaaagtcc attgtatttt ccgtgagtga ctatggcaag ttgtatgttc ttgacgatgc
     3361 agaattcctt gggcgaatct gcgagtactt catgccccac gaaaagggta aaatacgcta
     3421 tcatacagtt tatgagaaag gctttcgagc atataatgac ctgcaaaaga agtgtgtcga
     3481 agcagtcctt gctttcgaag aaaaggtcgt aaaagcaaag aagatgtccg aaaaagaggg
     3541 cgcccattac attgacttca gagagatcct tgcccagaca atgtgtaagg aagccgagaa
     3601 aactgctgtg aataaggtaa ggagagcctt tttccatcac cacctcaagt tcgtcataga
     3661 cgaatttgga ttgttttctg atgtgatgaa aaaatatggg attgagaaag aatggaagtt
     3721 tccagtgaag cccaagaaaa aaagaaaagt atcctcctac ccatacgatg ttccagatta
     3781 cgcttgacaa ctttattata catagttgga attcctagag ctcgctgatc agcctcgact
     3841 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3901 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3961 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4021 gaagagaata gcaggcatgc tggggagggc cgcaggaacc cctagtgatg gagttggcca
     4081 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4141 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     4201 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4261 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4321 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     4381 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4441 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4501 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4561 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     4621 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     4681 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4741 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4801 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4861 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4921 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4981 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5041 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5101 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5161 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5221 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5281 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5341 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5401 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5461 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5521 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5581 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5641 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5701 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5761 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5821 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5881 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5941 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6001 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6061 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6121 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6181 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6241 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6301 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6361 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6421 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6481 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6541 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6601 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6661 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6781 tgctcacatg t
//
