LOCUS       sequence-455913-        6650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid AAV vector expressing Cas13bt3 and hU6-driven expression of 
            guide RNAs. Contains SapI sites for guide cloning flanked by 5' and 
            3' full-length DRs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6650)
  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 6650)
  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: pC178: pAAV.U6.SapI.CMV.Cas13bt3
FEATURES             Location/Qualifiers
     source          1..6650
                     /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    597..621
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        682..985
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        986..1189
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1140..1160
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1211..1235)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1244..1264
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3587..3607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3614..3640
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3644..3668)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3698..3905
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3913..4053
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3913..4042
                     /label=AAV2 ITR
     rep_origin      4128..4583
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4215..4234)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4425..4446
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4600..4619)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4719..4741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4779..4797)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4865..4969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4970..5038,5039..5830)
                     /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(5188..5207)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6001..6589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6490..6509
                     /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 cagcagaaga gcgagctctt ctgctggagc
      481 agcccccgat ttgtggggtg attacagctt tttttgaatt ctgatgcggt gaagctttag
      541 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcaa
      601 gtttgtacaa aaaagcaggc ttagttatta atagtaatca attacggggt cattagttca
      661 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      721 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      781 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      841 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      901 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      961 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     1021 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     1081 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     1141 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     1201 ccgtcagatc acccagcttt cttgtacaaa gtgggccacc atgccaaaga agaaacggaa
     1261 ggtcgctcag gtctctaagc agacatccaa gaagagggaa ctgtccattg atgaatatca
     1321 gggtgccaga aaatggtgtt ttacaatcgc ctttaataag gccttggtca atcgggacaa
     1381 gaatgacgga ttattcgttg aatcactact ccgccacgag aaatactcca agcatgattg
     1441 gtacgacgag gacaccaggg cattgatcaa atgtagcacg caagccgcaa atgctaaggc
     1501 ggaggctctg cgaaactact tctcacatta caggcacagt ccagggtgcc tgaccttcac
     1561 cgcggaggac gagttgcgga ctattatgga gcgggcatac gagcgggcca tattcgagtg
     1621 tagacgacgc gaaaccgaag tcattattga gttcccctca ctgttcgagg gcgacaggat
     1681 aactacagct ggggtagtct tttttgtgtc tttcttcgtg gaaagacgtg tgctcgaccg
     1741 tctctacgga gccgtgagcg gtctgaaaaa gaatgagggc caatacaaac tcacccggaa
     1801 ggcactctcg atgtactgtc taaaggattc ccgatttacc aaggcatggg acaaaagggt
     1861 gctactgttc agggatatcc tggcacagct ggggcggatt cccgccgagg catatgaata
     1921 ctaccacggc gaacagggcg ataaaaaacg cgctaatgat aacgagggca caaatccaaa
     1981 gcgccataaa gacaaattca ttgaattcgc attgcattac cttgaagccc agcacagcga
     2041 aatctgcttc ggtcggagac acatcgtacg tgaggaggct ggcgctggag atgagcataa
     2101 gaagcaccgc accaagggga aggtagttgt cgattttagt aagaaagatg aagaccagtc
     2161 atattacatc tccaagaaca acgtgattgt gcgcattgac aagaacgccg gaccccgctc
     2221 ttatagaatg gggctgaatg agctcaagta cctggtcctg cttagcctcc agggaaaagg
     2281 ggatgacgct attgctaagc tctatcgcta cagacagcac gttgagaaca tcctcgacgt
     2341 tgtgaaagtt acagataagg ataaccatgt gttcctgccg agatttgtgt tagaacagca
     2401 cggcatcggc aggaaggctt tcaagcaaag gatcgatgga agagtcaagc atgtgcgtgg
     2461 agtgtgggaa aaaaaaaaag ctgccactaa cgagatgacc ctgcacgaga aagctaggga
     2521 tatccttcag tatgtaaacg agaactgtac tcgtagcttt aaccctggag aatacaatag
     2581 actgttagtg tgccttgttg ggaaggatgt ggagaacttt caggccggcc ttaagcgact
     2641 ccagttagct gagcggatag atggacgagt gtatagtatc ttcgcgcaga cttctacgat
     2701 caatgagatg catcaagtcg tgtgtgacca aattttaaac aggctgtgca ggatcggcga
     2761 tcaaaagctg tatgactatg tcgggctggg taaaaaggat gaaatagatt acaaacagaa
     2821 ggtcgcctgg tttaaggagc acatctcgat cagacggggg tttttgcgga agaaattttg
     2881 gtacgattct aaaaaaggat ttgcaaagct ggtggaggag catctggaga gcggaggcgg
     2941 gcagcgcgac gtaggtcttg ataagaaata ctaccacata gacgcgattg gccggttcga
     3001 gggcgcgaac cctgccctgt atgaaaccct cgcccgagac agattgtgct tgatgatggc
     3061 acagtatttt ctgggttcag tacggaagga acttggtaac aagatcgtgt ggagcaatga
     3121 cagcatcgaa ttgcctgttg aagggagtgt gggtaatgaa aaatccattg tgttttccgt
     3181 gagtgactat ggcaaactgt atgttctgga cgacgcggaa ttccttggga ggatctgcga
     3241 gtacttcatg ccccacgaaa agggcaaaat acgctatcat acagtttatg agaaaggctt
     3301 tcgagcatat aatgacctgc aaaagaagtg cgtcgaagca gtgctagctt tcgaagagaa
     3361 ggtcgttaaa gcaaagaaga tgtccgagaa agagggcgcc cattacattg acttcagaga
     3421 gatcctggct cagacaatgt gcaaggaagc cgagaaaact gctgtgaata aagtaaggag
     3481 agcctttttc catcaccacc tcaagttcgt catagacgaa tttggactct tttctgatgt
     3541 gatgaaaaaa tatgggattg agaaagagtg gaagttccca gtgaagccca agaaaaaaag
     3601 aaaagtatcc tcctacccat acgatgttcc agattacgct tgaacccagc tttcttgtac
     3661 aaagtgggaa ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     3721 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3781 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3841 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     3901 ggggagggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3961 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4021 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4081 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4141 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4201 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4261 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4321 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4381 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4441 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     4501 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4561 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     4621 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4681 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4741 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     4801 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4861 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4921 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4981 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5041 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5101 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5161 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5221 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5281 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5341 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5401 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5461 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5521 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5581 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5641 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     5701 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5761 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5821 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5881 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5941 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6001 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6061 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
