LOCUS       sequence-394038-        5573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tdKatushka2 under the TRE promoter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5573)
  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 5573)
  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-tdKatushka2-WPRE
FEATURES             Location/Qualifiers
     source          1..5573
                     /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"
     promoter        652..965
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     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"
     primer_bind     938..962
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(989..1005)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(989..1005)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1026..1730
                     /codon_start=1
                     /product="improved version of the oligomeric far-red 
                     fluorescent protein TurboFP635/Katushka (Luker et al., 
                     2015)"
                     /label=Katushka2S
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             1026..1730
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             1800..2504
                     /codon_start=1
                     /product="improved version of the oligomeric far-red 
                     fluorescent protein TurboFP635/Katushka (Luker et al., 
                     2015)"
                     /label=Katushka2S
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     CDS             1800..2504
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="MVGEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTM
                     KIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGG
                     VLTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHAQ
                     MALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAV
                     ARYCDLPSKLGHS"
     misc_feature    2541..3129
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2594..2614)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3012..3023)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3290..3371
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3327..3346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3385..3405)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3385..3403)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3424..3440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3424..3440)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3437..3459)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3448..3464
                     /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(3472..3478,3479..3496,3497..3502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3517..3538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3655..3672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3826..4414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3906..3925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4585..5376,5377..5445))
                     /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     5208..5227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5446..5550)
                     /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 tagtttactc
      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 aattcctgca gcccggggga tccactagtt ctagatttaa gaaggagata
     1021 tacatatggt gggtgaggat agcgtgctga tcaccgagaa catgcacatg aaactgtaca
     1081 tggagggcac cgtgaacgac caccacttca agtgcacatc cgagggcgaa ggcaagccct
     1141 acgagggcac ccagaccatg aagatcaagg tggtcgaggg cggccctctc cccttcgcct
     1201 tcgacatcct ggctaccagc ttcatgtacg gcagcaaaac ctttatcaac cacacccagg
     1261 gcatccccga cttctttaag cagtccttcc ctgagggctt cacatgggag aggatcacca
     1321 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag aacggctgcc
     1381 tcatctacaa cgtcaagatc aacggggtga acttcccatc caacggccct gtgatgcaga
     1441 agaaaacact cggctgggag gccagcaccg agatgctgta ccccgctgac agcggcctga
     1501 gaggccatgc ccagatggcc ctgaagctcg tgggcggggg ctacctgcac tgctccctca
     1561 agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc ttctacttcg
     1621 tggacaggag actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg
     1681 agatggctgt ggccaggtac tgcgacctgc ctagcaaact ggggcacagc gggcatggca
     1741 ccggcagcac cggcagcggc agctccggta ccgcctcctc cgaggacaac aacatggcca
     1801 tggtgggtga ggatagcgtg ctgatcaccg agaacatgca catgaaactg tacatggagg
     1861 gcaccgtgaa cgaccaccac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
     1921 gcacccagac catgaagatc aaggtggtcg agggcggccc tctccccttc gccttcgaca
     1981 tcctggctac cagcttcatg tacggcagca aaacctttat caaccacacc cagggcatcc
     2041 ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagaggatc accacatacg
     2101 aagacggggg cgtgctgacc gctacccagg acaccagcct ccagaacggc tgcctcatct
     2161 acaacgtcaa gatcaacggg gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa
     2221 cactcggctg ggaggccagc accgagatgc tgtaccccgc tgacagcggc ctgagaggcc
     2281 atgcccagat ggccctgaag ctcgtgggcg ggggctacct gcactgctcc ctcaagacca
     2341 catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcttctac ttcgtggaca
     2401 ggagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag cacgagatgg
     2461 ctgtggccag gtactgcgac ctgcctagca aactggggca cagctccgga ctcagatctt
     2521 gagcggccgc gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     2581 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2641 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2701 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2761 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2821 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2881 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     2941 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     3001 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3061 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3121 cctccccgca tcgataccgt cgacgcggcc gcatcgataa gcttgtcgac gatatctcta
     3181 gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3241 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     3301 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3361 tttttcactg cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     3421 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3481 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3541 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3601 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3661 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3721 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3781 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3841 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3901 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3961 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4021 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4081 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4141 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4201 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4261 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4321 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4381 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4441 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4501 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4561 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4621 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4681 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4741 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4801 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4861 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     4921 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4981 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5041 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5101 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5161 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5221 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5281 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5341 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5401 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5461 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5521 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
