LOCUS       sequence-115507-        5625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Mito-ONL in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5625)
  AUTHORS   Takai A, Nakano M, Saito K, Haruno R, Watanabe TM, Ohyanagi T, Jin 
            T, Okada Y, Nagai T
  TITLE     Expanded palette of Nano-lanterns for real-time multicolor 
            luminescence imaging.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Apr 7;112(14):4352-6. doi: 
            10.1073/pnas.1418468112. Epub 2015 Mar 23.
  PUBMED    25831507
REFERENCE   2  (bases 1 to 5625)
  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"; doi: 
            "10.1073/pnas.1418468112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-04-7- 7"; volume: "112"; issue: "14"; pages: "4352-6"
COMMENT     Sequence Label: pONL-N1_Mito
FEATURES             Location/Qualifiers
     source          1..5625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             597..671
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      699..708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             705..1358
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             705..1358
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    2413..2534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2450..2469)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2504..2523
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2541..2996)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2678..2699)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2890..2909
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3023..3127
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3129..3486
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3129..3320
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3152..3172)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3337..3472
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3521..4315
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3521..4315
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3575..3594)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4185..4204
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4503..4522)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4547..4594
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4923..5511
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5412..5431
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgt
      601 ccgtcctgac gccgctgctg ctgcggggct tgacaggctc ggcccggcgg ctcccagtgc
      661 cgcgcgccaa gatccattcg ttgggggatc caccggtcgc caccatggtg agcgtgatca
      721 agcccgagat gaagatgagg tactacatgg acggctccgt caatgggcat gagttcacaa
      781 tcgagggtga gggcacaggc agaccttacg agggacatca ggagatgaca ctgcgcgtca
      841 caatggccga gggcgggcca atgcctttcg ccttcgacct ggtgtcccac gtgttctgtt
      901 acggccacag agtttttact aaatatccag aagagatccc agactatttc aagcaggcct
      961 ttcctgaggg cctgtcctgg gagaggtccc tggagttcga ggacggcggc tccgcctccg
     1021 tgagcgccca catcagcctg aggggcaaca ccttctacca caagtccaag ttcaccggcg
     1081 tgaacttccc cgccgacggc cccatcatgc agaaccagag cgtggactgg gagccctcca
     1141 ccgagaagat caccgccagc gacggcgtgc tgaagggcga cgtgaccatg tacctgaagc
     1201 tggagggcgg cggcaaccac aagtgccaga tgaagaccac ctacaaggcc gccaaggaga
     1261 tcctggagat gcccggcgac cactacatcg gccacaggct ggtgaggaag accgagggca
     1321 acatcaccga gcaggtggag gacgccgtgg cccactccgg taccaaggtg tacgaccccg
     1381 agcaacgcaa acgcatgatc actgggcctc agtggtgggc tcgctgcaag caaatgaacg
     1441 tgctggactc cttcatcaac tactatgatt ccgagaagca cgccgagaac gccgtgattt
     1501 ttctgcatgg taacgctacc tccagctacc tgtggaggca cgtcgtgcct cacatcgagc
     1561 ccgtggctag atgcatcatc cctgatctga tcggaatggg taagtccggc aagagcggga
     1621 atggctcata tcgcctcctg gatcactaca agtacctcac cgcttggttc gagctgctga
     1681 accttccaaa gaaaatcatc tttgtgggcc acgactgggg gagcgctctg gcctttcact
     1741 acgcctacga gcaccaagac aggatcaagg ccatcgtcca tatggagagt gtcgtggacg
     1801 tgatcgagtc ctggatgggg tggcctgaca tcgaggagga gctggccctg atcaagagcg
     1861 aagagggcga gaaaatggtg cttgagaata acttcttcgt cgagaccctg ttgccaagca
     1921 agatcatgcg gaaactggag cctgaggagt tcgctgccta cctggagcca ttcaaggaga
     1981 agggcgaggt tagacggcct accctctcct ggcctcgcga gatccctctc gttaagggag
     2041 gcaagcccga cgtcgtccag attgtccgca actacaacgc ctaccttcgg gccagcgacg
     2101 atctgcctaa gctgttcatc gagtccgacc ctgggttctt ttccaacgct attgtcgagg
     2161 gagctaagaa gttccctaac accgagttcg tgaaggtgaa gggcctccac ttcctccagg
     2221 aggacgctcc agatgaaatg ggtaagtaca tcaagagctt cgtggagcgc gtgctgaaga
     2281 acgagcagta ggcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     2341 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     2401 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2461 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2521 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     2581 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2641 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2701 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2761 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2821 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2881 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2941 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     3001 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3061 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3121 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     3181 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3241 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3301 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3361 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3421 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3481 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3541 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3601 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3661 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     3721 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3781 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3841 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3901 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3961 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4021 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     4081 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4141 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4201 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4261 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4321 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4381 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4441 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     4501 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     4561 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     4621 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     4681 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     4741 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     4801 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4861 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4921 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4981 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5041 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     5101 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5161 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5221 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5281 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5341 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5401 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5461 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5521 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5581 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
