LOCUS       sequence-255174-        5095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kusabira- Orange-CENP-A expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5095)
  AUTHORS   Sugimoto K, Senda-Murata K, Oka S
  TITLE     Construction of three quadruple-fluorescent MDA435 cell lines that 
            enable monitoring of the whole chromosome segregation process in the
            living state.
  JOURNAL   Mutat Res. 2008 Nov 17;657(1):56-62. doi: 
            10.1016/j.mrgentox.2008.08.005. Epub 2008 Aug 19.
  PUBMED    18778791
REFERENCE   2  (bases 1 to 5095)
  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: 
            "10.1016/j.mrgentox.2008.08.005"; journalName: "Mutat Res"; date: 
            "2008-11-17- 17"; volume: "657"; issue: "1"; pages: "56-62"
COMMENT     Sequence Label: pmKO-AF8
FEATURES             Location/Qualifiers
     source          1..5095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      540..549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             546..1199
                     /codon_start=1
                     /product="monomeric Kusabira-Orange fluorescent protein 
                     (Karasawa et al., 2004)"
                     /label=mKO
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAKGGPMPFAFDLVSHVFCYGHRPFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQFKTTYKAAKKILKMPGSHYISHRLVRKTEGNITELVEDAVAHS"
     polyA_signal    1824..1945
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1861..1880)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1915..1934
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1952..2407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2089..2110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2301..2320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2434..2538
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2540..2897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2540..2731
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2563..2583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2748..2883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2931..3725
                     /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             2931..3725
                     /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(2985..3004)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3595..3614
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3913..3932)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3957..4004
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4333..4921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4822..4841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcattg
      541 ccaccatggt gagtgtgatt aaaccagaga tgaagatgag gtactacatg gacggctccg
      601 tcaatgggca tgagttcaca attgaaggtg aaggcacagg cagaccttac gagggacatc
      661 aagagatgac actacgcgtc acaatggcca agggcgggcc aatgcctttc gcgtttgact
      721 tagtgtcaca cgtgttctgt tacggccaca gaccttttac taaatatcca gaagagatac
      781 cagactattt caaacaagca tttcctgaag gcctgtcatg ggaaaggtcg ttggagttcg
      841 aagatggtgg gtccgcttca gtcagtgcgc atataagcct tagaggaaac accttctacc
      901 acaaatccaa atttactggg gttaactttc ctgccgatgg tcctatcatg caaaaccaaa
      961 gtgttgattg ggagccatca accgagaaaa ttactgccag cgacggagtt ctgaagggtg
     1021 atgttacgat gtacctaaaa cttgaaggag gcggcaatca caaatgccaa ttcaagacta
     1081 cttacaaggc ggcaaaaaag attcttaaaa tgccaggaag ccattacatc agccatcgcc
     1141 tcgtcaggaa aaccgaaggc aacattactg agctggtaga agatgcagta gctcattccg
     1201 gatctcgagc tctgcggcgt gtcatgggcc cgcgccgccg gagccgaaag cccgaggccc
     1261 cgaggaggcg cagcccgagc ccgaccccga cccccggccc ctcccggcgg ggcccctcct
     1321 taggcgcttc ctcccatcaa cacagtcggc ggagacaagg ttggctaaag gagatccgaa
     1381 agcttcagaa gagcacacac ctcttgataa ggaagctgcc cttcagccgc ctggcaagag
     1441 aaatatgtgt taaattcact cgtggtgtgg acttcaattg gcaagcccag gccctattgg
     1501 ccctacaaga ggcagcagaa gcatttctag ttcatctctt tgaggacgcc tatctcctca
     1561 ccttacatgc aggccgagtt actctcttcc caaaggatgt gcaactggcc cggaggatcc
     1621 ggggccttga ggagggactc ggctcgacgg taccggaact gcagcagaga attcgggaaa
     1681 ctcgagaaca aagcttgaat aagcggccgc gactctagat cataatcagc cataccacat
     1741 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1801 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     1861 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1921 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     1981 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2041 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2101 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2161 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2221 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2281 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2341 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2401 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2461 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2521 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2581 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2641 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2701 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2761 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2821 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2881 gcctaggctt ttgcaaagat catcaagaga caggatgagg atcgtttcgc atgattgaac
     2941 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3001 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3061 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     3121 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3181 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3241 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3301 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3361 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3421 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     3481 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3541 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3601 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3661 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3721 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     3781 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3841 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     3901 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     3961 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     4021 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     4081 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     4141 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     4201 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4261 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4321 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4381 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4441 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4501 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4561 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4621 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4681 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4741 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4801 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4861 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4921 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4981 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt
     5041 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccg
//
