LOCUS       sequence-394333-        4815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of mRuby3 under the TRE promoter in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4815)
  AUTHORS   Leiwe MN, Fujimoto S, Baba T, Moriyasu D, Saha B, Sakaguchi R, 
            Inagaki S, Imai T
  TITLE     Automated neuronal reconstruction with super-multicolour Tetbow 
            labelling and threshold-based clustering of colour hues.
  JOURNAL   Nat Commun. 2024 Jun 25;15(1):5279. doi: 10.1038/s41467-024-49455-y.
  PUBMED    38918382
REFERENCE   2  (bases 1 to 4815)
  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: "Nat 
            Commun."; date: "2024-06-25"; volume: "15(1):5279. doi"; pages: " 
            10.1038/s41467-024-49455-y"
COMMENT     Sequence Label: pAAV-TRE-mRuby3-WPRE
FEATURES             Location/Qualifiers
     source          1..4815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   16..145
                     /label=AAV2 ITR
     protein_bind    219..237
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    255..273
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    290..308
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    326..344
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    362..380
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    397..415
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    433..451
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        463..501
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     498..522
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      536..545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             542..1252
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     misc_feature    1268..1856
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1321..1341)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1739..1750)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1859..1875)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1860..1876)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    1905..2026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1916..1935)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1970..1989
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2188..2317
                     /label=AAV2 ITR
     rep_origin      complement(2554..3142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2634..2653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3313..4104,4105..4173))
                     /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     3936..3955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4174..4278)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4304..4759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4441..4462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4653..4672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcgctattac gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
       61 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
      121 gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct
      181 acgtagccat gctctaggaa gatctcgaat tggtttactc cctatcagtg atagagaacg
      241 tatgtcgagt ttactcccta tcagtgatag agaacgatgt cgagtttact ccctatcagt
      301 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      361 ctccctatca gtgatagaga acgtatgtcg agtttatccc tatcagtgat agagaacgta
      421 tgtcgagttt actccctatc agtgatagag aacgtatgtc gaggtaggcg tgtacggtgg
      481 gaggcctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag aattcgccac
      541 catggtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
      601 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca gaccgtacga
      661 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
      721 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cggccgacat
      781 ccctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
      841 cgaagatggt ggagtcgtca ccgtcacgca ggacaccagc cttgaggatg gcgagctcgt
      901 ctacaacgtc aaggtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
      961 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagagg
     1021 atacactgac atcgcactga aagttgatgg tggtggccat ctgcactgca acttcgtgac
     1081 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtgtcc atgccgttga
     1141 tcaccgcctg gaaaggatcg aggagagtga caatgaaacc tacgtagtgc aacgcgaagt
     1201 ggcagttgcc aaatacagca accttggtgg tggcatggac gagctgtaca agtaaaagct
     1261 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     1321 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     1381 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     1441 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     1501 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     1561 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     1621 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     1681 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     1741 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     1801 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     1861 ataccgtcga cctcgacccg ggcggccgct tcgagcagac atgataagat acattgatga
     1921 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1981 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2041 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa
     2101 cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac gtagataagt
     2161 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct
     2221 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     2281 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa tcggccaacg
     2341 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2401 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2461 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3421 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3541 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3601 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3721 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3841 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3961 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4021 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4141 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4201 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4261 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     4321 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4381 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4441 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4501 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4561 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4621 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4681 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4741 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4801 tcggtgcggg cctct
//
