LOCUS       sequence-442998-        7013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone for dSaCas9 (endonuclease dead Cas9 from Staphylococcus
            aureus) and Sa-gRNA Scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7013)
  AUTHORS   Kantor B, Odonovan B, Rittiner J, Hodgson D, Lindner N, Guerrero S, 
            Dong W, Zhang A, Chiba-Falek O
  TITLE     All-in-one AAV-delivered epigenome-editing platform: 
            proof-of-concept and therapeutic implications for neurodegenerative 
            disorders.
  JOURNAL   bioRxiv [Preprint]. 2024 May 19:2023.04.14.536951. doi: 
            10.1101/2023.04.14.536951.
  PUBMED    38798630
REFERENCE   2  (bases 1 to 7013)
  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: "bioRxiv 
            [Preprint]. 2024 May 19:2023.04.14.536951. doi: 
            10.1101/2023.04.14.536951."
COMMENT     Sequence Label: pBK1290-AAV-dSaCas9
FEATURES             Location/Qualifiers
     source          1..7013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        186..397
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      405..414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             414..479
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             489..509
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             513..533
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3714..3761
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3828..3848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3858..3906
                     /note="synthetic polyadenylation signal"
     promoter        3918..4166
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3918..3938
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4089..4108
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4187..4262
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4276..4416
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4276..4405
                     /label=AAV2 ITR
     rep_origin      4491..4946
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4578..4597)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4788..4809
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4963..4982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5082..5104
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5142..5160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5228..5332
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5333..5401,5402..6193)
                     /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(5551..5570)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6364..6952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6853..6872
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg
      181 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      241 ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      301 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      361 cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggtgccacc atggactaca
      421 aagaccatga cggtgattat aaagatcatg acatagatta caaggatgac gatgacaagg
      481 gtggatcgcc aaagaaaaag aggaaagtcg ccccaaagaa gaagcggaag gtcggtatcc
      541 acggagtccc agcagccaag cggaactaca tcctgggcct ggccatcggc atcaccagcg
      601 tgggctacgg catcatcgac tacgagacac gggacgtgat cgatgccggc gtgcggctgt
      661 tcaaagaggc caacgtggaa aacaacgagg gcaggcggag caagagaggc gccagaaggc
      721 tgaagcggcg gaggcggcat agaatccaga gagtgaagaa gctgctgttc gactacaacc
      781 tgctgaccga ccacagcgag ctgagcggca tcaaccccta cgaggccaga gtgaagggcc
      841 tgagccagaa gctgagcgag gaagagttct ctgccgccct gctgcacctg gccaagagaa
      901 gaggcgtgca caacgtgaac gaggtggaag aggacaccgg caacgagctg tccaccaaag
      961 agcagatcag ccggaacagc aaggccctgg aagagaaata cgtggccgaa ctgcagctgg
     1021 aacggctgaa gaaagacggc gaagtgcggg gcagcatcaa cagattcaag accagcgact
     1081 acgtgaaaga agccaaacag ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga
     1141 gcttcatcga cacctacatc gacctgctgg aaacccggcg gacctactat gagggacctg
     1201 gcgagggcag ccccttcggc tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc
     1261 actgcaccta cttccccgag gaactgcgga gcgtgaagta cgcctacaac gccgacctgt
     1321 acaacgccct gaacgacctg aacaatctcg tgatcaccag ggacgagaac gagaagctgg
     1381 aatattacga gaagttccag atcatcgaga acgtgttcaa gcagaagaag aagcccaccc
     1441 tgaagcagat cgccaaagaa atcctcgtga acgaagagga tattaagggc tacagagtga
     1501 ccagcaccgg caagcccgag ttcaccaacc tgaaggtgta ccacgacatc aaggacatta
     1561 ccgcccggaa agagattatt gagaacgccg agctgctgga tcagattgcc aagatcctga
     1621 ccatctacca gagcagcgag gacatccagg aagaactgac caatctgaac tccgagctga
     1681 cccaggaaga gatcgagcag atctctaatc tgaagggcta taccggcacc cacaacctga
     1741 gcctgaaggc catcaacctg atcctggacg agctgtggca caccaacgac aaccagatcg
     1801 ctatcttcaa ccggctgaag ctggtgccca agaaggtgga cctgtcccag cagaaagaga
     1861 tccccaccac cctggtggac gacttcatcc tgagccccgt cgtgaagaga agcttcatcc
     1921 agagcatcaa agtgatcaac gccatcatca agaagtacgg cctgcccaac gacatcatta
     1981 tcgagctggc ccgcgagaag aactccaagg acgcccagaa aatgatcaac gagatgcaga
     2041 agcggaaccg gcagaccaac gagcggatcg aggaaatcat ccggaccacc ggcaaagaga
     2101 acgccaagta cctgatcgag aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt
     2161 acagcctgga agccatccct ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg
     2221 accacatcat ccccagaagc gtgtccttcg acaacagctt caacaacaag gtgctcgtga
     2281 agcaggaaga agccagcaag aagggcaacc ggaccccatt ccagtacctg agcagcagcg
     2341 acagcaagat cagctacgaa accttcaaga agcacatcct gaatctggcc aagggcaagg
     2401 gcagaatcag caagaccaag aaagagtatc tgctggaaga acgggacatc aacaggttct
     2461 ccgtgcagaa agacttcatc aaccggaacc tggtggatac cagatacgcc accagaggcc
     2521 tgatgaacct gctgcggagc tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca
     2581 tcaatggcgg cttcaccagc tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca
     2641 aggggtacaa gcaccacgcc gaggacgccc tgatcattgc caacgccgat ttcatcttca
     2701 aagagtggaa gaaactggac aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa
     2761 agcaggccga gagcatgccc gagatcgaaa ccgagcagga gtacaaagag atcttcatca
     2821 ccccccacca gatcaagcac attaaggact tcaaggacta caagtacagc caccgggtgg
     2881 acaagaagcc taatagagag ctgattaacg acaccctgta ctccacccgg aaggacgaca
     2941 agggcaacac cctgatcgtg aacaatctga acggcctgta cgacaaggac aatgacaagc
     3001 tgaaaaagct gatcaacaag agccccgaaa agctgctgat gtaccaccac gacccccaga
     3061 cctaccagaa actgaagctg attatggaac agtacggcga cgagaagaat cccctgtaca
     3121 agtactacga ggaaaccggg aactacctga ccaagtactc caaaaaggac aacggccccg
     3181 tgatcaagaa gattaagtat tacggcaaca aactgaacgc ccatctggac atcaccgacg
     3241 actaccccaa cagcagaaac aaggtcgtga agctgtccct gaagccctac agattcgacg
     3301 tgtacctgga caatggcgtg tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa
     3361 aagaaaacta ctacgaagtg aatagcaagt gctatgagga agctaagaag ctgaagaaga
     3421 tcagcaacca ggccgagttt atcgcctcct tctacaacaa cgatctgatc aagatcaacg
     3481 gcgagctgta tagagtgatc ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca
     3541 tgatcgacat cacctaccgc gagtacctgg aaaacatgaa cgacaagagg ccccccagga
     3601 tcattaagac aatcgcctcc aagacccaga gcattaagaa gtacagcaca gacattctgg
     3661 gcaacctgta tgaagtgaaa tctaagaagc accctcagat catcaaaaag ggcaaaaggc
     3721 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggatccggt ggaggaagtg
     3781 gcgggtcagg gtcgggtggc actagtatag ctagcggagg tggttcgcca aagaagaaac
     3841 ggaaggtggg atcctgaaat aaaagatctt tgttttcatt agatctgtgt gttggttttt
     3901 tgtgtgcggc cggtaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     3961 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     4021 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     4081 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     4141 atatcttgtg gaaaggacga aacaccggag accacggcag gtctcagttt tagtactctg
     4201 gaaacagaat ctactaaaac aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga
     4261 gatttttgcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4321 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4381 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4441 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4501 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4561 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4621 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4681 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4741 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4801 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     4861 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4921 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     4981 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5041 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5101 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5161 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5221 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5281 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5341 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5401 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5461 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5521 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5581 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5641 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5701 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5761 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5821 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5881 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5941 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6001 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     6061 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6121 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6181 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6241 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6301 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6361 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6421 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6481 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6541 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6601 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6661 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6721 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6781 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6841 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6901 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6961 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
