LOCUS       sequence-354789-        4808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AcGFP1 with mitochondria-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4808)
  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 4808)
  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: pAcGFP1-mito
FEATURES             Location/Qualifiers
     source          1..4808
                     /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             597..671
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      699..708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             717..1436
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    1596..1717
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1633..1652)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1687..1706
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1724..2179)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1861..1882)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2073..2092
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2206..2310
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2312..2669
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2312..2503
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2335..2355)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2520..2655
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2704..3498
                     /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             2704..3498
                     /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(2758..2777)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3368..3387
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3686..3705)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3730..3777
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4106..4694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4595..4614
                     /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 gctagcatgt
      601 ccgtcctgac gccgctgctg ctgcggggct tgacaggctc ggcccggcgg ctcccagtgc
      661 cgcgcgccaa gatccattcg ttgggggatc caccggtcgc caccatggcc acaaccatgg
      721 tgagcaaggg cgccgagctg ttcaccggca tcgtgcccat cctgatcgag ctgaatggcg
      781 atgtgaatgg ccacaagttc agcgtgagcg gcgagggcga gggcgatgcc acctacggca
      841 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc tgtgccctgg cccaccctgg
      901 tgaccaccct gagctacggc gtgcagtgct tctcacgcta ccccgatcac atgaagcagc
      961 acgacttctt caagagcgcc atgcctgagg gctacatcca ggagcgcacc atcttcttcg
     1021 aggatgacgg caactacaag tcgcgcgccg aggtgaagtt cgagggcgat accctggtga
     1081 atcgcatcga gctgaccggc accgatttca aggaggatgg caacatcctg ggcaataaga
     1141 tggagtacaa ctacaacgcc cacaatgtgt acatcatgac cgacaaggcc aagaatggca
     1201 tcaaggtgaa cttcaagatc cgccacaaca tcgaggatgg cagcgtgcag ctggccgacc
     1261 actaccagca gaataccccc atcggcgatg gccctgtgct gctgcccgat aaccactacc
     1321 tgtccaccca gagcgccctg tccaaggacc ccaacgagaa gcgcgatcac atgatctact
     1381 tcggcttcgt gaccgccgcc gccatcaccc acggcatgga tgagctgtac aagtagcggc
     1441 cgaagggcga attctgcaga tatccatcac actggcggcc gcgactctag atcataatca
     1501 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     1561 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     1621 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     1681 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat
     1741 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     1801 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     1861 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     1921 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     1981 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2041 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     2101 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     2161 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2221 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2281 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     2341 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2401 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2461 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     2521 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     2581 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     2641 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     2701 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     2761 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     2821 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     2881 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     2941 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3001 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3061 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3121 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3181 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     3241 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3301 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3361 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3421 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3481 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     3541 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3601 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3661 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     3721 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     3781 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     3841 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     3901 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     3961 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4021 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4081 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4141 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4201 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4261 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4321 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4381 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4441 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4501 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4561 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4621 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4681 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4741 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4801 ccatgcat
//
