LOCUS       sequence-353619-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of a catalytically inactive SaCas9 (dSaCas9) fused 
            with three copies of SunTag sequence from Synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  AUTHORS   Yamamoto N, Morita S, Hatada I, Ideta-Otsuka M, Tamura H, Narita M, 
            Igarashi K
  TITLE     Engineering of AAV-Mediated in Vivo Targeted DNA Methylation Editing
            System via Staphylococcus Aureus Derived Cas9-SunTag
  JOURNAL   Research Square [Preprint] 2021. doi: 10.21203/rs.3.rs-1065603/v1.
REFERENCE   2  (bases 1 to 7369)
  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: "Research 
            Square [Preprint] 2021. doi: 10.21203/rs.3.rs-1065603/v1."
COMMENT     Sequence Label: AAV-hSyn-dSaCas9-NLS-3xHA-3xSunTag_bGHpA
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(821..1612,1613..1681))
                     /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     1444..1463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1682..1786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1854..1872
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1910..1932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2032..2051
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2068..2523)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2205..2226)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2417..2436
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2598..2738
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2609..2738
                     /label=AAV2 ITR
     promoter        2753..3200
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      3207..3216
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3219..3239
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6420..6467
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6474..6554
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             6618..6674
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6741..6797
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6864..6920
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     primer_bind     complement(7014..7031)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7020..7227
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7240..7369
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
      901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
      961 ggcttccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1801 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1861 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1921 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1981 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     2041 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     2101 cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc
     2161 cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga
     2221 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2281 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     2341 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2401 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg
     2461 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2521 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2581 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     2641 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     2701 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcctcg agagtgcaag
     2761 tgggttttag gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc
     2821 cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga
     2881 ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc
     2941 cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt
     3001 cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg
     3061 ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc
     3121 tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg
     3181 tgtcgtgcct gagagcgcag accggtgcca ccatggcccc aaagaagaag cggaaggtcg
     3241 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggcc atcggcatca
     3301 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc
     3361 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca
     3421 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact
     3481 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga
     3541 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca
     3601 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca
     3661 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc
     3721 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca
     3781 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg
     3841 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg
     3901 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga
     3961 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg
     4021 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga
     4081 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc
     4141 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca
     4201 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg
     4261 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga
     4321 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg
     4381 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca
     4441 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc
     4501 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga
     4561 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct
     4621 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca
     4681 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga
     4741 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca
     4801 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt
     4861 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg
     4921 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc
     4981 tcgtgaagca ggaagaagcc agcaagaagg gcaaccggac cccattccag tacctgagca
     5041 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg
     5101 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca
     5161 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca
     5221 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga
     5281 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc
     5341 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca
     5401 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg
     5461 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct
     5521 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc
     5581 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg
     5641 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg
     5701 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc
     5761 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc
     5821 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg
     5881 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca
     5941 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat
     6001 tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga
     6061 tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga
     6121 agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga
     6181 tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag
     6241 tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc
     6301 ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca
     6361 ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggca
     6421 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tcctacccat
     6481 acgatgttcc agattacgct tatccctacg acgtgcctga ttatgcatac ccatatgatg
     6541 tccccgacta tgccgacggc attggtagtg ggagcaacgg cagcagcgga tccaacggtc
     6601 cgactgacgc cgcggaagag gagcttctga gcaaaaacta tcacctcgaa aacgaggttg
     6661 cgcgactgaa gaaaggaagc gggtccggtg gaagtggctc cggatctgga ggttctggca
     6721 gcggaggtag cggcagtggc gaagagctcc ttagtaagaa ctatcatctg gaaaatgagg
     6781 tagcgcgctt aaagaaaggg tcgggaagtg gcggcagcgg aagtgggagt ggagggagcg
     6841 gttctggcgg ttccggcagt ggagaggagt tgctgtctaa gaactaccac ttagaaaacg
     6901 aagtcgcacg gctaaaaaaa ggttccggct ccggcggctc cggttctgga agcgggggct
     6961 cgggatcagg tggatctgga tcaggataag aattcctaga gctcgctgat cagcctcgac
     7021 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     7081 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     7141 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     7201 ggaagagaat agcaggcatg ctggggagct agaggccgca ggaaccccta gtgatggagt
     7261 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     7321 gacgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcagg
//
