LOCUS       sequence-353669-        6190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Chimeric guide for SaCas9 and reverse 
            tetracycline-controlled transactivator for doxycycline controlled 
            expression from Synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6190)
  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 6190)
  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-TetRC-2A-Tomato-bGHpA;U6_BsaI-sgRNA
FEATURES             Location/Qualifiers
     source          1..6190
                     /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        149..596
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             join(609..1226,1227..1232,1233..1268,1269..1271,1272..1307,
                     1308..1310,1311..1346,1347..1352)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1362..1418
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1419..2849
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     primer_bind     complement(2874..2891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2880..3087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(3099..3174)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(3195..3443)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3253..3272)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3423..3443)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   3453..3593
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3453..3582
                     /label=AAV2 ITR
     rep_origin      3668..4123
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3755..3774)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3965..3986
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4140..4159)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4259..4281
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4319..4337)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4405..4509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4510..4578,4579..5370)
                     /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(4728..4747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5541..6129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6030..6049
                     /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 gactcgagag tgcaagtggg ttttaggacc aggatgaggc
      181 ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca
      241 ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg
      301 cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
      361 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc
      421 cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg
      481 cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
      541 cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagaccg
      601 gtgccaccat gtctagactg gacaagagca aagtcataaa cggagctctg gaattactca
      661 atggtgtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc
      721 agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg
      781 agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact
      841 ttctgcggaa caacgccaag tcataccgct gtgctctcct ctcacatcgc gacggggcta
      901 aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg
      961 cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg
     1021 gccactttac actgggctgc gtattggagg aacaggagca tcaagtagca aaagaggaaa
     1081 gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg
     1141 accggcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg
     1201 agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc ccttgacgat tttgacttag
     1261 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc
     1321 ttgacgattt tgaccttgac atgctccccg ggggaagcgg agctactaac ttcagcctgc
     1381 tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggtgagcaag ggcgaggagg
     1441 tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt
     1501 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     1561 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttcatgt
     1621 acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag aagctgtcct
     1681 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg
     1741 tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag atgcgcggca
     1801 ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     1861 ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag gccctgaagc
     1921 tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc aagaagcccg
     1981 tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc cacaacgagg
     2041 actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg ttcctggggc
     2101 atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag gacaacaaca
     2161 tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc
     2221 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2281 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt
     2341 tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc
     2401 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg
     2461 tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc
     2521 gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2581 cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc
     2641 tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac atggccaaga
     2701 agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca
     2761 acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc
     2821 tgtacggcat ggacgagctg tacaagtgag aattcctaga gctcgctgat cagcctcgac
     2881 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2941 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3001 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3061 ggaagagaat agcaggcatg ctggggaggt accaaaaatc tcgccaacaa gttgacgaga
     3121 taaacacggc attttgcctt gttttagtag attctgtttc cagagtacta aaactgagac
     3181 ctgccgtggt ctccggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     3241 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     3301 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     3361 acagtcaaat taattccaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     3421 ggaatcatgg gaaataggcc ctcagcggcc gcaggaaccc ctagtgatgg agttggccac
     3481 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     3541 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     3601 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     3661 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3721 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     3781 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3841 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     3901 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3961 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     4021 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     4081 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4141 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4201 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4261 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4321 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4381 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4441 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4501 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4561 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4621 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4681 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4741 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4801 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4861 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4921 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4981 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5041 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5101 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5161 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5221 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5281 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5341 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5401 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5461 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5521 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5581 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5641 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5701 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5761 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5821 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5881 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5941 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6001 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6061 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6121 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6181 gctcacatgt
//
