LOCUS       sequence-394157-        4746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mRuby3 under the TRE promoter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4746)
  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 4746)
  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: pBS-TRE-mRuby3-WPRE
FEATURES             Location/Qualifiers
     source          1..4746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    659..677
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    695..713
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    730..748
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    766..784
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    802..820
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    837..855
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    873..891
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        903..941
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     938..962
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      976..985
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             982..1692
                     /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    1714..2302
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1767..1787)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2185..2196)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2463..2544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2500..2519)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2558..2578)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2558..2576)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2597..2613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2597..2613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2610..2632)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2621..2637
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2645..2651,2652..2669,2670..2675))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2690..2711
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2828..2845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2999..3587)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3079..3098)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3758..4549,4550..4618))
                     /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     4381..4400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4619..4723)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggtttactc
      661 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgatgt
      721 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
      781 gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttatccc
      841 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc
      901 gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga
      961 tcgcctggag aattcgccac catggtgtct aagggcgaag agctgatcaa ggaaaatatg
     1021 cgtatgaagg tggtcatgga aggttcggtc aacggccacc aattcaaatg cacaggtgaa
     1081 ggagaaggca gaccgtacga gggaactcaa accatgagga tcaaagtcat cgagggagga
     1141 cccctgccat ttgcctttga cattcttgcc acgtcgttca tgtatggcag ccgtactttt
     1201 atcaagtacc cggccgacat ccctgatttc tttaaacagt cctttcctga gggttttact
     1261 tgggaaagag ttacgagata cgaagatggt ggagtcgtca ccgtcacgca ggacaccagc
     1321 cttgaggatg gcgagctcgt ctacaacgtc aaggtcagag gggtaaactt tccctccaat
     1381 ggtcccgtga tgcagaagaa gaccaagggt tgggagccta atacagagat gatgtatcca
     1441 gcagatggtg gtctgagagg atacactgac atcgcactga aagttgatgg tggtggccat
     1501 ctgcactgca acttcgtgac aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg
     1561 cccggtgtcc atgccgttga tcaccgcctg gaaaggatcg aggagagtga caatgaaacc
     1621 tacgtagtgc aacgcgaagt ggcagttgcc aaatacagca accttggtgg tggcatggac
     1681 gagctgtaca agtaagcggc cgcgcttatc gataatcaac ctctggatta caaaatttgt
     1741 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     1801 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     1861 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     1921 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     1981 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     2041 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     2101 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     2161 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     2221 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     2281 tccctttggg ccgcctcccc gcatcgatac cgtcgacgcg gccgcatcga taagcttgtc
     2341 gacgatatct ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     2401 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     2461 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2521 caaataaagc atttttttca ctgcctagct tttgttccct ttagtgaggg ttaattgcgc
     2581 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2641 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2701 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2761 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     2821 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2881 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2941 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3001 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3061 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3121 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3181 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3241 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3301 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3361 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3421 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3481 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3541 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3601 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3661 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3721 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3781 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3841 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3901 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3961 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4021 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4081 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4141 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4201 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4261 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4321 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4381 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4441 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4501 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4561 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4621 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4681 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4741 tgccac
//
