LOCUS       sequence-111642-        5599 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses Mito-ONL in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5599)
  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 5599)
  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..5599
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     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"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             679..1332
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             679..1332
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    2387..2508
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2424..2443)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2478..2497
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2515..2970)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2652..2673)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2864..2883
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2997..3101
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3103..3460
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3103..3294
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3126..3146)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3311..3446
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3495..4289
                     /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             3495..4289
                     /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(3549..3568)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4159..4178
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4477..4496)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4521..4568
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4897..5485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5386..5405
                     /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 gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat ggtgagcgtg atcaagcccg agatgaagat gaggtactac
      721 atggacggct ccgtcaatgg gcatgagttc acaatcgagg gtgagggcac aggcagacct
      781 tacgagggac atcaggagat gacactgcgc gtcacaatgg ccgagggcgg gccaatgcct
      841 ttcgccttcg acctggtgtc ccacgtgttc tgttacggcc acagagtttt tactaaatat
      901 ccagaagaga tcccagacta tttcaagcag gcctttcctg agggcctgtc ctgggagagg
      961 tccctggagt tcgaggacgg cggctccgcc tccgtgagcg cccacatcag cctgaggggc
     1021 aacaccttct accacaagtc caagttcacc ggcgtgaact tccccgccga cggccccatc
     1081 atgcagaacc agagcgtgga ctgggagccc tccaccgaga agatcaccgc cagcgacggc
     1141 gtgctgaagg gcgacgtgac catgtacctg aagctggagg gcggcggcaa ccacaagtgc
     1201 cagatgaaga ccacctacaa ggccgccaag gagatcctgg agatgcccgg cgaccactac
     1261 atcggccaca ggctggtgag gaagaccgag ggcaacatca ccgagcaggt ggaggacgcc
     1321 gtggcccact ccggtaccaa ggtgtacgac cccgagcaac gcaaacgcat gatcactggg
     1381 cctcagtggt gggctcgctg caagcaaatg aacgtgctgg actccttcat caactactat
     1441 gattccgaga agcacgccga gaacgccgtg atttttctgc atggtaacgc tacctccagc
     1501 tacctgtgga ggcacgtcgt gcctcacatc gagcccgtgg ctagatgcat catccctgat
     1561 ctgatcggaa tgggtaagtc cggcaagagc gggaatggct catatcgcct cctggatcac
     1621 tacaagtacc tcaccgcttg gttcgagctg ctgaaccttc caaagaaaat catctttgtg
     1681 ggccacgact gggggagcgc tctggccttt cactacgcct acgagcacca agacaggatc
     1741 aaggccatcg tccatatgga gagtgtcgtg gacgtgatcg agtcctggat ggggtggcct
     1801 gacatcgagg aggagctggc cctgatcaag agcgaagagg gcgagaaaat ggtgcttgag
     1861 aataacttct tcgtcgagac cctgttgcca agcaagatca tgcggaaact ggagcctgag
     1921 gagttcgctg cctacctgga gccattcaag gagaagggcg aggttagacg gcctaccctc
     1981 tcctggcctc gcgagatccc tctcgttaag ggaggcaagc ccgacgtcgt ccagattgtc
     2041 cgcaactaca acgcctacct tcgggccagc gacgatctgc ctaagctgtt catcgagtcc
     2101 gaccctgggt tcttttccaa cgctattgtc gagggagcta agaagttccc taacaccgag
     2161 ttcgtgaagg tgaagggcct ccacttcctc caggaggacg ctccagatga aatgggtaag
     2221 tacatcaaga gcttcgtgga gcgcgtgctg aagaacgagc agtaggcggc cgcgactcta
     2281 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     2341 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     2401 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2461 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     2521 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2581 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2641 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2701 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2761 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2821 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2881 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2941 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     3001 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3061 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     3121 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3181 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3241 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3301 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3361 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3421 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     3481 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3541 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3601 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3661 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3721 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3781 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3841 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3901 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3961 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4021 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4081 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4141 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4201 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4261 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4321 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4381 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4441 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     4501 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     4561 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     4621 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4681 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     4741 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4801 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4861 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4921 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4981 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5041 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5101 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5161 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5221 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     5281 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     5341 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5401 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5461 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5521 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5581 ccgtattacc gccatgcat
//
