LOCUS       sequence-353664-        6343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Chimeric guide for SaCas9 and reverse 
            tetracycline-controlled transactivator for doxycycline controlled 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6343)
  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 6343)
  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-TetRC-2A-Tomato-bGHpA;U6_BsaI-sgRNA
FEATURES             Location/Qualifiers
     source          1..6343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(762..1379,1380..1385,1386..1421,1422..1424,1425..1460,
                     1461..1463,1464..1499,1500..1505)
                     /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             1515..1571
                     /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             1572..3002
                     /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(3027..3044)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3033..3240
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(3252..3327)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(3348..3596)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3406..3425)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3576..3596)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   3606..3746
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3606..3735
                     /label=AAV2 ITR
     rep_origin      3821..4276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3908..3927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4118..4139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4293..4312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4412..4434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4472..4490)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4558..4662
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4663..4731,4732..5523)
                     /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(4881..4900)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5694..6282
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6183..6202
                     /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 gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catgtctaga ctggacaaga
      781 gcaaagtcat aaacggagct ctggaattac tcaatggtgt cggtatcgaa ggcctgacga
      841 caaggaaact cgctcaaaag ctgggagttg agcagcctac cctgtactgg cacgtgaaga
      901 acaagcgggc cctgctcgat gccctgccaa tcgagatgct ggacaggcat catacccact
      961 tctgccccct ggaaggcgag tcatggcaag actttctgcg gaacaacgcc aagtcatacc
     1021 gctgtgctct cctctcacat cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag
     1081 agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc
     1141 tggagaacgc actgtacgct ctgtccgccg tgggccactt tacactgggc tgcgtattgg
     1201 aggaacagga gcatcaagta gcaaaagagg aaagagagac acctaccacc gattctatgc
     1261 ccccacttct gagacaagca attgagctgt tcgaccggca gggagccgaa cctgccttcc
     1321 ttttcggcct ggaactaatc atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg
     1381 ggccgaccga cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg
     1441 actttgacct tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc
     1501 ccgggggaag cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga
     1561 accctggacc tatggtgagc aagggcgagg aggtcatcaa agagttcatg cgcttcaagg
     1621 tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1681 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct
     1741 tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc
     1801 ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg
     1861 tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg
     1921 gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa
     1981 tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg
     2041 tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg
     2101 agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg
     2161 acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc
     2221 gctccgaggg ccgccaccac ctgttcctgg ggcatggcac cggcagcacc ggcagcggca
     2281 gctccggcac cgcctcctcc gaggacaaca acatggccgt catcaaagag ttcatgcgct
     2341 tcaaggtgcg catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
     2401 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc
     2461 tgcccttcgc ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga
     2521 agcaccccgc cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg
     2581 agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc
     2641 aggacggcac gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc
     2701 ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg
     2761 acggcgtgct gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc
     2821 tggtggagtt caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact
     2881 acgtggacac caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     2941 acgagcgctc cgagggccgc caccacctgt tcctgtacgg catggacgag ctgtacaagt
     3001 gagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3061 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3121 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3181 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     3241 ggtaccaaaa atctcgccaa caagttgacg agataaacac ggcattttgc cttgttttag
     3301 tagattctgt ttccagagta ctaaaactga gacctgccgt ggtctccggt gtttcgtcct
     3361 ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga
     3421 tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta
     3481 cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc aattatctct
     3541 ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcagcg
     3601 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3661 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3721 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3781 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3841 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     3901 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3961 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4021 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4081 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4141 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4201 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4261 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4321 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4381 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4441 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     4501 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4561 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4621 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4681 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4741 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4801 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4861 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4921 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4981 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5041 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5101 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5161 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5221 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5281 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5341 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     5401 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5461 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5521 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5581 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5641 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5701 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5761 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5821 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5881 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5941 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6001 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6061 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6121 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6181 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6241 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6301 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
