LOCUS       sequence-354828-        4892 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express mKate2 with mitochondria-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4892)
  AUTHORS   Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P, 
            Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y
  TITLE     Folding Latency of Fluorescent Proteins Affects the Mitochondrial 
            Localization of Fusion Proteins.
  JOURNAL   Cell Struct Funct. 2019 Dec 26;44(2):183-194. doi: 
            10.1247/csf.19028. Epub 2019 Nov 15.
  PUBMED    31735740
REFERENCE   2  (bases 1 to 4892)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1247/csf.19028";
            journalName: "Cell Struct Funct"; date: "2019-12-26- 26"; volume: 
            "44"; issue: "2"; pages: "183-194"
COMMENT     Sequence Label: pFX-mito-mKate2
FEATURES             Location/Qualifiers
     source          1..4892
                     /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             624..698
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             756..1454
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     polyA_signal    1680..1801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1717..1736)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1771..1790
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1808..2263)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1945..1966)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2157..2176
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2290..2394
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2396..2753
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2396..2587
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2419..2439)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2604..2739
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2788..3582
                     /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             2788..3582
                     /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(2842..2861)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3452..3471
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3770..3789)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3814..3861
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4190..4778
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4679..4698
                     /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 ccggttctag agccatgctc gagatgtccg tcctgacgcc gctgctgctg cggggcttga
      661 caggctcggc ccggcggctc ccagtgccgc gcgccaagat ccattcgttg ggggatccac
      721 cggtcgccac ctgcggccgc ggaggctccg gaggaatggt gagcgagctg attaaggaga
      781 acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat
      841 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gcggtcgagg
      901 gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcaaaa
      961 ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc cccgagggct
     1021 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     1081 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcccat
     1141 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggcctccacc gagaccctgt
     1201 accccgctga cggcggcctg gaaggcagag ccgacatggc cctgaagctc gtgggcgggg
     1261 gccacctgat ctgcaacttg aagaccacat acagatccaa gaaacccgct aagaacctca
     1321 agatgcccgg cgtctactat gtggacagaa gactggaaag aatcaaggag gccgacaaag
     1381 agacctacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac
     1441 tggggcacag atagatctct gactgactga gatgcgcgga tgtcgaacgg cagttcggcc
     1501 gaataggccg acagccggcc gtcggccgac gacacggcca gggccggcgg ggatccaccg
     1561 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     1621 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     1681 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     1741 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     1801 aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     1861 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     1921 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     1981 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     2041 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     2101 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2161 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     2221 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     2281 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2341 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     2401 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2461 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2521 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2581 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2641 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     2701 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     2761 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2821 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2881 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2941 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     3001 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3061 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3121 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3181 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3241 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3301 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3361 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3421 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3481 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3541 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3601 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3661 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3721 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     3781 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     3841 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     3901 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     3961 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     4021 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4081 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4141 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4201 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4261 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4321 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4381 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4441 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4501 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4561 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4621 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4681 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4741 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4801 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4861 attctgtgga taaccgtatt accgccatgc at
//
