LOCUS       sequence-354821-        4903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eqFP650 with mitochondria-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4903)
  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 4903)
  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: "10.1247/csf.19028";
            journalName: "Cell Struct Funct"; date: "2019-12-26- 26"; volume: 
            "44"; issue: "2"; pages: "183-194"
COMMENT     Sequence Label: pFX-mito-eqFP650
FEATURES             Location/Qualifiers
     source          1..4903
                     /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             762..1463
                     /codon_start=1
                     /product="near-infrared fluorescent protein eqFP670"
                     /label=NirFP
                     /note="mammalian codon-optimized"
                     /translation="MGEDSELISENMHMKLYMEGTVNGHHFKCTSEGEGKPYEGTQTAK
                     IKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGGV
                     LTATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHSQM
                     ALKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRKLERIKEADKETYVEQHEMAVA
                     RYCDLPSKLGHS"
     polyA_signal    1691..1812
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1728..1747)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1782..1801
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1819..2274)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1956..1977)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2168..2187
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2301..2405
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2407..2764
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2407..2598
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2430..2450)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2615..2750
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2799..3593
                     /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             2799..3593
                     /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(2853..2872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3463..3482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3781..3800)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3825..3872
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4201..4789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4690..4709
                     /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 ggaggatccg gaggagaatt catgggagag gatagcgagc
      781 tgatctccga gaacatgcac atgaaactgt acatggaggg caccgtgaac ggccaccact
      841 tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc gctaagatca
      901 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt
      961 acggcagcaa aacctttatc aaccacaccc agggcatccc cgacttcttt aagcagtcct
     1021 tccctgaggg cttcacatgg gagaggatca ccacatacga agacgggggc gtgctgaccg
     1081 ctacccagga caccagcctc cagaacggct gcctcatcta caacgtcaag atcaacgggg
     1141 tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccagca
     1201 ccgagatgct gtaccccgct gacagcggcc tgagaggcca tagtcagatg gccctgaagc
     1261 tcgtgggcgg gggctacctg cactgctccc tcaagaccac atacagatcc aagaaacccg
     1321 ctaagaacct caagatgccc ggcttctact tcgtggacag gaaactggaa agaatcaagg
     1381 aggccgacaa agagacctac gtcgagcagc acgagatggc tgtggccagg tactgcgacc
     1441 tgcctagcaa actggggcac agcagatctc tgactgactg agatgcgcgg atgtcgaacg
     1501 gcagttcggc cgaataggcc gacagccggc cgtcggccga cgacacggcc agggccggcg
     1561 gggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     1621 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     1681 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1741 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1801 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     1861 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     1921 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     1981 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2041 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2101 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2161 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2221 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     2281 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2341 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2401 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2461 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2521 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2581 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     2641 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     2701 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     2761 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2821 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2881 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2941 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     3001 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3061 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3121 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3181 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3241 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3301 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     3361 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3421 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3481 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3541 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3601 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3661 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3721 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     3781 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     3841 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     3901 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     3961 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     4021 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     4081 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4141 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4201 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4261 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4321 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4381 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4441 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4501 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4561 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4621 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4681 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4741 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4801 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4861 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
