LOCUS       sequence-352726-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of a catalytically inactive SaCas9 (dSaCas9) fused 
            with three copies of SunTag sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  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 7522)
  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-CMV-dSaCas9-NLS-3xHA-3xSunTag_bGHpA
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        60..439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        440..643
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     594..614
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      662..671
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             674..694
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3875..3922
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3929..4009
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             4073..4129
                     /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             4196..4252
                     /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             4319..4375
                     /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(4469..4486)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4475..4682
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4695..4824
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4886..5474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4966..4985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5645..6436,6437..6505))
                     /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     6268..6287
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6506..6610)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6678..6696
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6734..6756)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6856..6875
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6892..7347)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7029..7050)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7241..7260
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   join(7422..7522,1..40)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7433..7522,1..40)
                     /label=AAV2 ITR
ORIGIN
        1 cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccgcct cgaggcgttg
       61 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      121 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      181 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      481 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      541 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      601 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactaccgg
      661 tgccaccatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccaa
      721 gcggaactac atcctgggcc tggccatcgg catcaccagc gtgggctacg gcatcatcga
      781 ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg ttcaaagagg ccaacgtgga
      841 aaacaacgag ggcaggcgga gcaagagagg cgccagaagg ctgaagcggc ggaggcggca
      901 tagaatccag agagtgaaga agctgctgtt cgactacaac ctgctgaccg accacagcga
      961 gctgagcggc atcaacccct acgaggccag agtgaagggc ctgagccaga agctgagcga
     1021 ggaagagttc tctgccgccc tgctgcacct ggccaagaga agaggcgtgc acaacgtgaa
     1081 cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa gagcagatca gccggaacag
     1141 caaggccctg gaagagaaat acgtggccga actgcagctg gaacggctga agaaagacgg
     1201 cgaagtgcgg ggcagcatca acagattcaa gaccagcgac tacgtgaaag aagccaaaca
     1261 gctgctgaag gtgcagaagg cctaccacca gctggaccag agcttcatcg acacctacat
     1321 cgacctgctg gaaacccggc ggacctacta tgagggacct ggcgagggca gccccttcgg
     1381 ctggaaggac atcaaagaat ggtacgagat gctgatgggc cactgcacct acttccccga
     1441 ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg tacaacgccc tgaacgacct
     1501 gaacaatctc gtgatcacca gggacgagaa cgagaagctg gaatattacg agaagttcca
     1561 gatcatcgag aacgtgttca agcagaagaa gaagcccacc ctgaagcaga tcgccaaaga
     1621 aatcctcgtg aacgaagagg atattaaggg ctacagagtg accagcaccg gcaagcccga
     1681 gttcaccaac ctgaaggtgt accacgacat caaggacatt accgcccgga aagagattat
     1741 tgagaacgcc gagctgctgg atcagattgc caagatcctg accatctacc agagcagcga
     1801 ggacatccag gaagaactga ccaatctgaa ctccgagctg acccaggaag agatcgagca
     1861 gatctctaat ctgaagggct ataccggcac ccacaacctg agcctgaagg ccatcaacct
     1921 gatcctggac gagctgtggc acaccaacga caaccagatc gctatcttca accggctgaa
     1981 gctggtgccc aagaaggtgg acctgtccca gcagaaagag atccccacca ccctggtgga
     2041 cgacttcatc ctgagccccg tcgtgaagag aagcttcatc cagagcatca aagtgatcaa
     2101 cgccatcatc aagaagtacg gcctgcccaa cgacatcatt atcgagctgg cccgcgagaa
     2161 gaactccaag gacgcccaga aaatgatcaa cgagatgcag aagcggaacc ggcagaccaa
     2221 cgagcggatc gaggaaatca tccggaccac cggcaaagag aacgccaagt acctgatcga
     2281 gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg tacagcctgg aagccatccc
     2341 tctggaagat ctgctgaaca accccttcaa ctatgaggtg gaccacatca tccccagaag
     2401 cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg aagcaggaag aagccagcaa
     2461 gaagggcaac cggaccccat tccagtacct gagcagcagc gacagcaaga tcagctacga
     2521 aaccttcaag aagcacatcc tgaatctggc caagggcaag ggcagaatca gcaagaccaa
     2581 gaaagagtat ctgctggaag aacgggacat caacaggttc tccgtgcaga aagacttcat
     2641 caaccggaac ctggtggata ccagatacgc caccagaggc ctgatgaacc tgctgcggag
     2701 ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc atcaatggcg gcttcaccag
     2761 ctttctgcgg cggaagtgga agtttaagaa agagcggaac aaggggtaca agcaccacgc
     2821 cgaggacgcc ctgatcattg ccaacgccga tttcatcttc aaagagtgga agaaactgga
     2881 caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa aagcaggccg agagcatgcc
     2941 cgagatcgaa accgagcagg agtacaaaga gatcttcatc accccccacc agatcaagca
     3001 cattaaggac ttcaaggact acaagtacag ccaccgggtg gacaagaagc ctaatagaga
     3061 gctgattaac gacaccctgt actccacccg gaaggacgac aagggcaaca ccctgatcgt
     3121 gaacaatctg aacggcctgt acgacaagga caatgacaag ctgaaaaagc tgatcaacaa
     3181 gagccccgaa aagctgctga tgtaccacca cgacccccag acctaccaga aactgaagct
     3241 gattatggaa cagtacggcg acgagaagaa tcccctgtac aagtactacg aggaaaccgg
     3301 gaactacctg accaagtact ccaaaaagga caacggcccc gtgatcaaga agattaagta
     3361 ttacggcaac aaactgaacg cccatctgga catcaccgac gactacccca acagcagaaa
     3421 caaggtcgtg aagctgtccc tgaagcccta cagattcgac gtgtacctgg acaatggcgt
     3481 gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa aaagaaaact actacgaagt
     3541 gaatagcaag tgctatgagg aagctaagaa gctgaagaag atcagcaacc aggccgagtt
     3601 tatcgcctcc ttctacaaca acgatctgat caagatcaac ggcgagctgt atagagtgat
     3661 cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac atgatcgaca tcacctaccg
     3721 cgagtacctg gaaaacatga acgacaagag gccccccagg atcattaaga caatcgcctc
     3781 caagacccag agcattaaga agtacagcac agacattctg ggcaacctgt atgaagtgaa
     3841 atctaagaag caccctcaga tcatcaaaaa gggcaaaagg ccggcggcca cgaaaaaggc
     3901 cggccaggca aaaaagaaaa agggatccta cccatacgat gttccagatt acgcttatcc
     3961 ctacgacgtg cctgattatg catacccata tgatgtcccc gactatgccg acggcattgg
     4021 tagtgggagc aacggcagca gcggatccaa cggtccgact gacgccgcgg aagaggagct
     4081 tctgagcaaa aactatcacc tcgaaaacga ggttgcgcga ctgaagaaag gaagcgggtc
     4141 cggtggaagt ggctccggat ctggaggttc tggcagcgga ggtagcggca gtggcgaaga
     4201 gctccttagt aagaactatc atctggaaaa tgaggtagcg cgcttaaaga aagggtcggg
     4261 aagtggcggc agcggaagtg ggagtggagg gagcggttct ggcggttccg gcagtggaga
     4321 ggagttgctg tctaagaact accacttaga aaacgaagtc gcacggctaa aaaaaggttc
     4381 cggctccggc ggctccggtt ctggaagcgg gggctcggga tcaggtggat ctggatcagg
     4441 ataagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     4501 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4561 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4621 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     4681 gagctagagg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4741 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg
     4801 agcgagcgcg cagctgcctg caggacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4861 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4921 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4981 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5041 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5101 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5161 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5221 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5281 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5341 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5401 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5461 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5521 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5581 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5641 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5701 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5761 gccccagtgc tgcaatgata ccgcggcttc cacgctcacc ggctccagat ttatcagcaa
     5821 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5881 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5941 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6001 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6061 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6121 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6181 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6241 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6301 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6361 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6421 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6481 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6541 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6601 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6661 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     6721 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     6781 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     6841 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa
     6901 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6961 aatagaccga aatcggcaaa atcccttata aatcaaaaga atagcccgag atagagttga
     7021 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     7081 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt
     7141 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     7201 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     7261 cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     7321 cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat
     7381 accgcacaga tgcgtaagga gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc
     7441 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc
     7501 ggcctcagtg agcgagcgag cg
//
