LOCUS       sequence-455906-        6578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active Cas13e with VEGFA mRNA targeting gRNA2.
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: pC368: pAAV.CMV-Cas13e-VEGFA sgRNA2
FEATURES             Location/Qualifiers
     source          1..6578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(2176..2196)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2206..2230)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2251..2454)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2280..2300)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2455..2758
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(2951..3199)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3009..3028)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3179..3199)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3200..3220)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     repeat_region   3227..3367
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3227..3356
                     /label=AAV2 ITR
     rep_origin      complement(3429..4017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3509..3528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4188..4979,4980..5048))
                     /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     4811..4830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5049..5153)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5221..5239
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5277..5299)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5399..5418
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5435..5890)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5572..5593)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5784..5803
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5965..6105
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5976..6105
                     /label=AAV2 ITR
     polyA_signal    6113..6320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6350..6374
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(6378..6404)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(6411..6431)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 cattgtctgg gcaaggatct ctctgaagtc aatgtaatgg gcgccctctt tttcggacat
       61 cttctttgct tttacgacct tttcttcgaa agcaaggact gcttcgacac acttcttttg
      121 caggtcatta tatgctcgaa agcctttctc ataaactgta tgatagcgta ttttaccctt
      181 ttcgtggggc atgaagtact cgcagattcg cccaaggaat tctgcatcgt caagaacata
      241 caacttgcca tagtcactca cggaaaatac aatggacttt tcattaccga cactcccttc
      301 aacaggcaat tcgatgctgt cattgctcca aacaatcttg ttaccaagtt ccttccgtac
      361 tgaacccaga aaatattgtg ccatcatcaa gcacaatcta tctcgggcga gggtttcata
      421 caaggcaggg tttgctccct cgaaccgccc aattgcgtct atgtggtagt atttcttatc
      481 aagacctacg tcgcgctgcc cgcctccgct ctccaaatgt tcttccacca gctttgcaaa
      541 tcctttttta gaatcgtacc aaaatttctt ccgcaaaaac ccccgtctga tggaaatgtg
      601 ctccttaaac caggcgacct tctgtttgta atctatttca tccttcttac ccagcccgac
      661 atagtcatag agcttttgat caccgatcct gcaaagcctg ttcaaaattt ggtcacaaac
      721 gacttgatgc atttcattga ttgtagaagt ctgtgcgaat atactataca ctcgtccatc
      781 tatccgttca gccaactgga gtcgcttaag gccggcttga aagttctcta catccttccc
      841 aacaaggcac acaagcagtc tattgtattc tccaggatta aaggagcgag tacagttttc
      901 gtttacatac tgaaggatat ccctagcttt ttcgtgcagg gtcatctcgt tagtggctgc
      961 tttttttttt tcccacactc cgcgcacatg cttgactctt ccatcgatcc tttgcttgaa
     1021 tgccttccta ccaatgccgt gctgttccaa cacaaatcta ggcaggaata catgattatc
     1081 cttatctgta actttcacaa cgtcgaggat attctcaacg tgttgtctat agcgatagag
     1141 cttagcaata gcgtcatccc cttttccctg gaggctaagc aagaccaagt acttgagctc
     1201 attcagcccc attctataag agcgtggtcc ggcgttctta tcaatgcgca caattacgtt
     1261 attcttggat atgtaatatg actggtcttc atctttctta ctaaaatcga caactacctt
     1321 ccccttggtg cggtgcttct tatgttcatc tccagcacca gcctcttctc gtacaatgtg
     1381 gcgcctaccg aagcagattt cgctgtgctg ggcttcaaga taatgcaatg cgaattcaat
     1441 gaatttgtct ttatggcgct ttggatttgt gccctcgtta tcattagcgc gttttttatc
     1501 accctgttcg ccgtggtagt attcatatgc ttcggcggga attcgcccca actgtgccag
     1561 gatatccctg aacaacaata cccttttgtc ccatgccttg gtaaatcggg aatccttaag
     1621 acagtacatg gagagtgcct ttcgggtgag tttgtattgg ccctcattct ttttcagacc
     1681 gctcacggct ccgtagagtc ggtcgagcac tcgtctttca acgaagaaag agacaaaaaa
     1741 gactacccca gctgtagtta tcctatcgcc ctcgaacagt gaggggaact caataatgac
     1801 ttctgtttcg cgtcgtctac attcgaatat ggccctttcg tatgcccgct ccataatagt
     1861 ccgcaactcg tcctctgcgg tgaaggtcaa gcaccctgga ctgtgcctgt aatgtgagaa
     1921 gtaatttcga agagcctctg ccttagcatt tgcggcttgt gtgctacatt tgatcaatgc
     1981 cctggtgtcc tcatcgtacc aatcatgctt ggagtatttt tcgtggcgga gaagggattc
     2041 aacgaaaagt ccgtcattct tgtcccgatt gaccaaggcc ttattaaagg cgattgtaaa
     2101 acaccatttt ctggcacctt gatattcatc aatggaaagt tccctcttct tggatgtctg
     2161 cttagagacc tgagcgacct tccgtttctt ctttggcatg gtggcagcct gcttttttgt
     2221 acaaacttgg atctgacggt tcactaaacc agctctgctt atatagacct cccaccgtac
     2281 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     2341 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     2401 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     2461 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     2521 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     2581 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     2641 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     2701 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     2761 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactaaa
     2821 gcttcaccgc atcagaattc aaaaaaagct gtaatcaccc cacaaatcgg gggctgctcc
     2881 agcgaaggag gagggcagaa tcatcacgaa gtggctgtaa tcaccccaca aatcgggggc
     2941 tgctccagcc ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact
     3001 ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact
     3061 acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag
     3121 tcaaattaat tccaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa
     3181 tcatgggaaa taggccctcc aacttttcta tacaaagttg tctagaagga acccctagtg
     3241 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     3301 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     3361 ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3421 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3481 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3541 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3601 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3661 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3721 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3781 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3841 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3901 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3961 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4021 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4081 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4141 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4201 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4261 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4321 ataccgcggc ttccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4381 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4441 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4501 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4561 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4621 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4681 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4741 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4801 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4861 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4921 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4981 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5041 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5101 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5161 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     5221 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     5281 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     5341 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     5401 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta
     5461 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac cgaaatcggc
     5521 aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt tccagtttgg
     5581 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5641 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     5701 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5761 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc taaggcgctg
     5821 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5881 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa
     5941 ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg cgctcgctcg ctcactgagg
     6001 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
     6061 gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cctccccagc
     6121 atgcctgcta ttctcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     6181 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     6241 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     6301 ctggcaacta gaaggcacag tcgaggctga tcagcgagct ctaggaattc ccactttgta
     6361 caagaaagct gggttcaagc gtaatctgga acatcgtatg ggtaggagga tacttttctt
     6421 tttttcttgg gcttcactgg aaacttccat tctttctcaa tcccatattt tttcatcaca
     6481 tcagaaaaca atccaaattc gtctatgacg aacttgaggt ggtgatggaa aaaggctctc
     6541 cttaccttat tcacagcagt tttctcggct tccttaca
//
