LOCUS       sequence-401491-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active and codon optimised Cas13e with VEGFA 
            gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  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 6662)
  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: pM156: pAAV.CMV-Cas13e (GenScript CO)-VEGFA sgRNA
FEATURES             Location/Qualifiers
     source          1..6662
                     /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    609..633
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        694..997
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        998..1201
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1152..1172
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1223..1247)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1256..1276
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3599..3619
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3626..3652
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3656..3680)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3710..3917
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3925..4065
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3925..4054
                     /label=AAV2 ITR
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4612..4631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4731..4753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4791..4809)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4877..4981
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4982..5050,5051..5842)
                     /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(5200..5219)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6013..6601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6502..6521
                     /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 tttttttgaa ttctgatgcg
      541 gtgaagcttt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      601 ggagttccgc aagtttgtac aaaaaagcag gcttagttat taatagtaat caattacggg
      661 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      721 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      781 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      841 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      901 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      961 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1021 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1081 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1141 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1201 tggtttagtg aaccgtcaga tcacccagct ttcttgtaca aagtgggcca ccatgccaaa
     1261 gaagaaacgg aaggtcgctc aggtctctaa gcagacatcc aagaagaggg aactgtccat
     1321 tgatgaatat cagggtgcca gaaaatggtg ttttacaatc gcctttaata aggccttggt
     1381 caatcgggac aagaatgacg gattattcgt tgaatcacta ctccgccacg agaaatactc
     1441 caagcatgat tggtacgacg aggacaccag ggcattgatc aaatgtagca cgcaagccgc
     1501 aaatgctaag gcggaggctc tgcgaaacta cttctcacat tacaggcaca gtccagggtg
     1561 cctgaccttc accgcggagg acgagttgcg gactattatg gagcgggcat acgagcgggc
     1621 catattcgag tgtagacgac gcgaaaccga agtcattatt gagttcccct cactgttcga
     1681 gggcgacagg ataactacag ctggggtagt cttttttgtg tctttcttcg tggaaagacg
     1741 tgtgctcgac cgtctctacg gagccgtgag cggtctgaaa aagaatgagg gccaatacaa
     1801 actcacccgg aaggcactct cgatgtactg tctaaaggat tcccgattta ccaaggcatg
     1861 ggacaaaagg gtgctactgt tcagggatat cctggcacag ctggggcgga ttcccgccga
     1921 ggcatatgaa tactaccacg gcgaacaggg cgataaaaaa cgcgctaatg ataacgaggg
     1981 cacaaatcca aagcgccata aagacaaatt cattgaattc gcattgcatt accttgaagc
     2041 ccagcacagc gaaatctgct tcggtcggag acacatcgta cgtgaggagg ctggcgctgg
     2101 agatgagcat aagaagcacc gcaccaaggg gaaggtagtt gtcgatttta gtaagaaaga
     2161 tgaagaccag tcatattaca tctccaagaa caacgtgatt gtgcgcattg acaagaacgc
     2221 cggaccccgc tcttatagaa tggggctgaa tgagctcaag tacctggtcc tgcttagcct
     2281 ccagggaaaa ggggatgacg ctattgctaa gctctatcgc tacagacagc acgttgagaa
     2341 catcctcgac gttgtgaaag ttacagataa ggataaccat gtgttcctgc cgagatttgt
     2401 gttagaacag cacggcatcg gcaggaaggc tttcaagcaa aggatcgatg gaagagtcaa
     2461 gcatgtgcgt ggagtgtggg aaaaaaaaaa agctgccact aacgagatga ccctgcacga
     2521 gaaagctagg gatatccttc agtatgtaaa cgagaactgt actcgtagct ttaaccctgg
     2581 agaatacaat agactgttag tgtgccttgt tgggaaggat gtggagaact ttcaggccgg
     2641 ccttaagcga ctccagttag ctgagcggat agatggacga gtgtatagta tcttcgcgca
     2701 gacttctacg atcaatgaga tgcatcaagt cgtgtgtgac caaattttaa acaggctgtg
     2761 caggatcggc gatcaaaagc tgtatgacta tgtcgggctg ggtaaaaagg atgaaataga
     2821 ttacaaacag aaggtcgcct ggtttaagga gcacatctcg atcagacggg ggtttttgcg
     2881 gaagaaattt tggtacgatt ctaaaaaagg atttgcaaag ctggtggagg agcatctgga
     2941 gagcggaggc gggcagcgcg acgtaggtct tgataagaaa tactaccaca tagacgcgat
     3001 tggccggttc gagggcgcga accctgccct gtatgaaacc ctcgcccgag acagattgtg
     3061 cttgatgatg gcacagtatt ttctgggttc agtacggaag gaacttggta acaagatcgt
     3121 gtggagcaat gacagcatcg aattgcctgt tgaagggagt gtgggtaatg aaaaatccat
     3181 tgtgttttcc gtgagtgact atggcaaact gtatgttctg gacgacgcgg aattccttgg
     3241 gaggatctgc gagtacttca tgccccacga aaagggcaaa atacgctatc atacagttta
     3301 tgagaaaggc tttcgagcat ataatgacct gcaaaagaag tgcgtcgaag cagtgctagc
     3361 tttcgaagag aaggtcgtta aagcaaagaa gatgtccgag aaagagggcg cccattacat
     3421 tgacttcaga gagatcctgg ctcagacaat gtgcaaggaa gccgagaaaa ctgctgtgaa
     3481 taaagtaagg agagcctttt tccatcacca cctcaagttc gtcatagacg aatttggact
     3541 cttttctgat gtgatgaaaa aatatgggat tgagaaagag tggaagttcc cagtgaagcc
     3601 caagaaaaaa agaaaagtat cctcctaccc atacgatgtt ccagattacg cttgaaccca
     3661 gctttcttgt acaaagtggg aattcctaga gctcgctgat cagcctcgac tgtgccttct
     3721 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3781 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3841 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     3901 agcaggcatg ctggggaggg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     3961 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4021 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4081 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4501 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4621 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4681 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4741 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4801 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4861 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4921 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4981 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5041 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5101 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5161 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5221 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5281 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5341 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5401 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5461 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5521 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5581 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5641 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     5701 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5761 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5821 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5881 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6661 gt
//
