LOCUS       sequence-116543-        5704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ITPKA-ONL in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5704)
  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 5704)
  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_ITPKA
FEATURES             Location/Qualifiers
     source          1..5704
                     /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"
     regulatory      778..787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             784..1437
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             784..1437
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    2492..2613
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2529..2548)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2583..2602
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2620..3075)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2757..2778)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2969..2988
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3102..3206
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3208..3565
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3208..3399
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3231..3251)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3416..3551
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3600..4394
                     /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             3600..4394
                     /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(3654..3673)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4264..4283
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4582..4601)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4626..4673
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5002..5590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5491..5510
                     /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 caagcttatg ggcatggcgc gaccacgggg cgcggggccc
      661 tgcagccccg ggttggagcg ggctccgcgc cggagcgtcg gggagctgcg cctgctcttc
      721 gaagcgcgct gcgccgcagt cgccgccgca gcggccgcag ggtcggatcc accggtcgcc
      781 accatggtga gcgtgatcaa gcccgagatg aagatgaggt actacatgga cggctccgtc
      841 aatgggcatg agttcacaat cgagggtgag ggcacaggca gaccttacga gggacatcag
      901 gagatgacac tgcgcgtcac aatggccgag ggcgggccaa tgcctttcgc cttcgacctg
      961 gtgtcccacg tgttctgtta cggccacaga gtttttacta aatatccaga agagatccca
     1021 gactatttca agcaggcctt tcctgagggc ctgtcctggg agaggtccct ggagttcgag
     1081 gacggcggct ccgcctccgt gagcgcccac atcagcctga ggggcaacac cttctaccac
     1141 aagtccaagt tcaccggcgt gaacttcccc gccgacggcc ccatcatgca gaaccagagc
     1201 gtggactggg agccctccac cgagaagatc accgccagcg acggcgtgct gaagggcgac
     1261 gtgaccatgt acctgaagct ggagggcggc ggcaaccaca agtgccagat gaagaccacc
     1321 tacaaggccg ccaaggagat cctggagatg cccggcgacc actacatcgg ccacaggctg
     1381 gtgaggaaga ccgagggcaa catcaccgag caggtggagg acgccgtggc ccactccggt
     1441 accaaggtgt acgaccccga gcaacgcaaa cgcatgatca ctgggcctca gtggtgggct
     1501 cgctgcaagc aaatgaacgt gctggactcc ttcatcaact actatgattc cgagaagcac
     1561 gccgagaacg ccgtgatttt tctgcatggt aacgctacct ccagctacct gtggaggcac
     1621 gtcgtgcctc acatcgagcc cgtggctaga tgcatcatcc ctgatctgat cggaatgggt
     1681 aagtccggca agagcgggaa tggctcatat cgcctcctgg atcactacaa gtacctcacc
     1741 gcttggttcg agctgctgaa ccttccaaag aaaatcatct ttgtgggcca cgactggggg
     1801 agcgctctgg cctttcacta cgcctacgag caccaagaca ggatcaaggc catcgtccat
     1861 atggagagtg tcgtggacgt gatcgagtcc tggatggggt ggcctgacat cgaggaggag
     1921 ctggccctga tcaagagcga agagggcgag aaaatggtgc ttgagaataa cttcttcgtc
     1981 gagaccctgt tgccaagcaa gatcatgcgg aaactggagc ctgaggagtt cgctgcctac
     2041 ctggagccat tcaaggagaa gggcgaggtt agacggccta ccctctcctg gcctcgcgag
     2101 atccctctcg ttaagggagg caagcccgac gtcgtccaga ttgtccgcaa ctacaacgcc
     2161 taccttcggg ccagcgacga tctgcctaag ctgttcatcg agtccgaccc tgggttcttt
     2221 tccaacgcta ttgtcgaggg agctaagaag ttccctaaca ccgagttcgt gaaggtgaag
     2281 ggcctccact tcctccagga ggacgctcca gatgaaatgg gtaagtacat caagagcttc
     2341 gtggagcgcg tgctgaagaa cgagcagtag gcggccgcga ctctagatca taatcagcca
     2401 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     2461 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2521 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2581 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     2641 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     2701 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2761 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2821 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2881 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2941 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3001 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3061 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3121 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3181 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     3241 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3301 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3361 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3421 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3481 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3541 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     3601 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3661 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3721 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3781 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3841 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3901 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3961 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4021 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4081 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     4141 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4201 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4261 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4321 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4381 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4441 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4501 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4561 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     4621 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     4681 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     4741 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     4801 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     4861 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4921 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4981 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5041 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5101 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5161 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5221 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5281 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5341 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5401 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5461 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5521 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5581 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5641 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     5701 gcat
//
