LOCUS       sequence-111645-        5629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NLS-ONL in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5629)
  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 5629)
  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_3xNLS
FEATURES             Location/Qualifiers
     source          1..5629
                     /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             610..630
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             634..654
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             658..678
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      703..712
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             709..1362
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             709..1362
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    2417..2538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2454..2473)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2508..2527
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2545..3000)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2682..2703)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2894..2913
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3027..3131
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3133..3490
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3133..3324
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3156..3176)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3341..3476
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3525..4319
                     /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             3525..4319
                     /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(3579..3598)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4189..4208
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4507..4526)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4551..4598
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4927..5515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5416..5435
                     /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 gctagccacc
      601 atgctggtac caaaaaagaa gagaaaggtt gtaccaaaaa agaagagaaa ggttgtacca
      661 aaaaagaaga gaaaggtttt cgaagggccg gatccaccgg tcgccaccat ggtgagcgtg
      721 atcaagcccg agatgaagat gaggtactac atggacggct ccgtcaatgg gcatgagttc
      781 acaatcgagg gtgagggcac aggcagacct tacgagggac atcaggagat gacactgcgc
      841 gtcacaatgg ccgagggcgg gccaatgcct ttcgccttcg acctggtgtc ccacgtgttc
      901 tgttacggcc acagagtttt tactaaatat ccagaagaga tcccagacta tttcaagcag
      961 gcctttcctg agggcctgtc ctgggagagg tccctggagt tcgaggacgg cggctccgcc
     1021 tccgtgagcg cccacatcag cctgaggggc aacaccttct accacaagtc caagttcacc
     1081 ggcgtgaact tccccgccga cggccccatc atgcagaacc agagcgtgga ctgggagccc
     1141 tccaccgaga agatcaccgc cagcgacggc gtgctgaagg gcgacgtgac catgtacctg
     1201 aagctggagg gcggcggcaa ccacaagtgc cagatgaaga ccacctacaa ggccgccaag
     1261 gagatcctgg agatgcccgg cgaccactac atcggccaca ggctggtgag gaagaccgag
     1321 ggcaacatca ccgagcaggt ggaggacgcc gtggcccact ccggtaccaa ggtgtacgac
     1381 cccgagcaac gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg
     1441 aacgtgctgg actccttcat caactactat gattccgaga agcacgccga gaacgccgtg
     1501 atttttctgc atggtaacgc tacctccagc tacctgtgga ggcacgtcgt gcctcacatc
     1561 gagcccgtgg ctagatgcat catccctgat ctgatcggaa tgggtaagtc cggcaagagc
     1621 gggaatggct catatcgcct cctggatcac tacaagtacc tcaccgcttg gttcgagctg
     1681 ctgaaccttc caaagaaaat catctttgtg ggccacgact gggggagcgc tctggccttt
     1741 cactacgcct acgagcacca agacaggatc aaggccatcg tccatatgga gagtgtcgtg
     1801 gacgtgatcg agtcctggat ggggtggcct gacatcgagg aggagctggc cctgatcaag
     1861 agcgaagagg gcgagaaaat ggtgcttgag aataacttct tcgtcgagac cctgttgcca
     1921 agcaagatca tgcggaaact ggagcctgag gagttcgctg cctacctgga gccattcaag
     1981 gagaagggcg aggttagacg gcctaccctc tcctggcctc gcgagatccc tctcgttaag
     2041 ggaggcaagc ccgacgtcgt ccagattgtc cgcaactaca acgcctacct tcgggccagc
     2101 gacgatctgc ctaagctgtt catcgagtcc gaccctgggt tcttttccaa cgctattgtc
     2161 gagggagcta agaagttccc taacaccgag ttcgtgaagg tgaagggcct ccacttcctc
     2221 caggaggacg ctccagatga aatgggtaag tacatcaaga gcttcgtgga gcgcgtgctg
     2281 aagaacgagc agtaggcggc cgcgactcta gatcataatc agccatacca catttgtaga
     2341 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     2401 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2461 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2521 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     2581 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2641 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     2701 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     2761 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2821 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2881 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2941 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3001 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3061 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3121 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3181 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3241 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3301 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3361 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3421 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3481 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3541 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3601 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3661 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     3721 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3781 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3841 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3901 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3961 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4021 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4081 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4141 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4201 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4261 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4321 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4381 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4441 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     4501 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     4561 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     4621 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     4681 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     4741 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     4801 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4861 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4921 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4981 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5041 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     5101 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5161 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5221 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5281 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5341 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5401 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5461 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5521 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5581 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
