LOCUS       sequence-353629-        7375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of a catalytically inactive SaCas9 (dSaCas9) fused 
            with three copies of SunTag sequence from doxycycine-inducible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7375)
  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 7375)
  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-TRE-dSaCas9-NLS-3xHA-3xSunTag_bGHpA
FEATURES             Location/Qualifiers
     source          1..7375
                     /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
     protein_bind    join(2782..2800,2801..2823,2824..2842,2843..2865,2866..2884,
                     2885..2907,2908..2926,2927..2949,2950..2968,2969..2991,
                     2992..3010,3011..3033,3034..3052)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        3085..3123
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3120..3144
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3213..3222
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3225..3245
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6426..6473
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6480..6560
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             6624..6680
                     /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             6747..6803
                     /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             6870..6926
                     /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(7020..7037)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7026..7233
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7246..7375
                     /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 agatccagtt
     2761 tggactagga tcctttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
     2821 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
     2881 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
     2941 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca
     3001 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa
     3061 agtcgagctc ggtacccggg tcgaggtagg cgtgtacggt gggaggccta tataagcaga
     3121 gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat
     3181 agaagacacc gggaccgatc cagcctaccg gtgccaccat ggccccaaag aagaagcgga
     3241 aggtcggtat ccacggagtc ccagcagcca agcggaacta catcctgggc ctggccatcg
     3301 gcatcaccag cgtgggctac ggcatcatcg actacgagac acgggacgtg atcgatgccg
     3361 gcgtgcggct gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg agcaagagag
     3421 gcgccagaag gctgaagcgg cggaggcggc atagaatcca gagagtgaag aagctgctgt
     3481 tcgactacaa cctgctgacc gaccacagcg agctgagcgg catcaacccc tacgaggcca
     3541 gagtgaaggg cctgagccag aagctgagcg aggaagagtt ctctgccgcc ctgctgcacc
     3601 tggccaagag aagaggcgtg cacaacgtga acgaggtgga agaggacacc ggcaacgagc
     3661 tgtccaccaa agagcagatc agccggaaca gcaaggccct ggaagagaaa tacgtggccg
     3721 aactgcagct ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc aacagattca
     3781 agaccagcga ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag gcctaccacc
     3841 agctggacca gagcttcatc gacacctaca tcgacctgct ggaaacccgg cggacctact
     3901 atgagggacc tggcgagggc agccccttcg gctggaagga catcaaagaa tggtacgaga
     3961 tgctgatggg ccactgcacc tacttccccg aggaactgcg gagcgtgaag tacgcctaca
     4021 acgccgacct gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc agggacgaga
     4081 acgagaagct ggaatattac gagaagttcc agatcatcga gaacgtgttc aagcagaaga
     4141 agaagcccac cctgaagcag atcgccaaag aaatcctcgt gaacgaagag gatattaagg
     4201 gctacagagt gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg taccacgaca
     4261 tcaaggacat taccgcccgg aaagagatta ttgagaacgc cgagctgctg gatcagattg
     4321 ccaagatcct gaccatctac cagagcagcg aggacatcca ggaagaactg accaatctga
     4381 actccgagct gacccaggaa gagatcgagc agatctctaa tctgaagggc tataccggca
     4441 cccacaacct gagcctgaag gccatcaacc tgatcctgga cgagctgtgg cacaccaacg
     4501 acaaccagat cgctatcttc aaccggctga agctggtgcc caagaaggtg gacctgtccc
     4561 agcagaaaga gatccccacc accctggtgg acgacttcat cctgagcccc gtcgtgaaga
     4621 gaagcttcat ccagagcatc aaagtgatca acgccatcat caagaagtac ggcctgccca
     4681 acgacatcat tatcgagctg gcccgcgaga agaactccaa ggacgcccag aaaatgatca
     4741 acgagatgca gaagcggaac cggcagacca acgagcggat cgaggaaatc atccggacca
     4801 ccggcaaaga gaacgccaag tacctgatcg agaagatcaa gctgcacgac atgcaggaag
     4861 gcaagtgcct gtacagcctg gaagccatcc ctctggaaga tctgctgaac aaccccttca
     4921 actatgaggt ggaccacatc atccccagaa gcgtgtcctt cgacaacagc ttcaacaaca
     4981 aggtgctcgt gaagcaggaa gaagccagca agaagggcaa ccggacccca ttccagtacc
     5041 tgagcagcag cgacagcaag atcagctacg aaaccttcaa gaagcacatc ctgaatctgg
     5101 ccaagggcaa gggcagaatc agcaagacca agaaagagta tctgctggaa gaacgggaca
     5161 tcaacaggtt ctccgtgcag aaagacttca tcaaccggaa cctggtggat accagatacg
     5221 ccaccagagg cctgatgaac ctgctgcgga gctacttcag agtgaacaac ctggacgtga
     5281 aagtgaagtc catcaatggc ggcttcacca gctttctgcg gcggaagtgg aagtttaaga
     5341 aagagcggaa caaggggtac aagcaccacg ccgaggacgc cctgatcatt gccaacgccg
     5401 atttcatctt caaagagtgg aagaaactgg acaaggccaa aaaagtgatg gaaaaccaga
     5461 tgttcgagga aaagcaggcc gagagcatgc ccgagatcga aaccgagcag gagtacaaag
     5521 agatcttcat caccccccac cagatcaagc acattaagga cttcaaggac tacaagtaca
     5581 gccaccgggt ggacaagaag cctaatagag agctgattaa cgacaccctg tactccaccc
     5641 ggaaggacga caagggcaac accctgatcg tgaacaatct gaacggcctg tacgacaagg
     5701 acaatgacaa gctgaaaaag ctgatcaaca agagccccga aaagctgctg atgtaccacc
     5761 acgaccccca gacctaccag aaactgaagc tgattatgga acagtacggc gacgagaaga
     5821 atcccctgta caagtactac gaggaaaccg ggaactacct gaccaagtac tccaaaaagg
     5881 acaacggccc cgtgatcaag aagattaagt attacggcaa caaactgaac gcccatctgg
     5941 acatcaccga cgactacccc aacagcagaa acaaggtcgt gaagctgtcc ctgaagccct
     6001 acagattcga cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg aagaatctgg
     6061 atgtgatcaa aaaagaaaac tactacgaag tgaatagcaa gtgctatgag gaagctaaga
     6121 agctgaagaa gatcagcaac caggccgagt ttatcgcctc cttctacaac aacgatctga
     6181 tcaagatcaa cggcgagctg tatagagtga tcggcgtgaa caacgacctg ctgaaccgga
     6241 tcgaagtgaa catgatcgac atcacctacc gcgagtacct ggaaaacatg aacgacaaga
     6301 ggccccccag gatcattaag acaatcgcct ccaagaccca gagcattaag aagtacagca
     6361 cagacattct gggcaacctg tatgaagtga aatctaagaa gcaccctcag atcatcaaaa
     6421 agggcaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatcct
     6481 acccatacga tgttccagat tacgcttatc cctacgacgt gcctgattat gcatacccat
     6541 atgatgtccc cgactatgcc gacggcattg gtagtgggag caacggcagc agcggatcca
     6601 acggtccgac tgacgccgcg gaagaggagc ttctgagcaa aaactatcac ctcgaaaacg
     6661 aggttgcgcg actgaagaaa ggaagcgggt ccggtggaag tggctccgga tctggaggtt
     6721 ctggcagcgg aggtagcggc agtggcgaag agctccttag taagaactat catctggaaa
     6781 atgaggtagc gcgcttaaag aaagggtcgg gaagtggcgg cagcggaagt gggagtggag
     6841 ggagcggttc tggcggttcc ggcagtggag aggagttgct gtctaagaac taccacttag
     6901 aaaacgaagt cgcacggcta aaaaaaggtt ccggctccgg cggctccggt tctggaagcg
     6961 ggggctcggg atcaggtgga tctggatcag gataagaatt cctagagctc gctgatcagc
     7021 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     7081 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     7141 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     7201 ggattgggaa gagaatagca ggcatgctgg ggagctagag gccgcaggaa cccctagtga
     7261 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     7321 tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcagg
//
