LOCUS       sequence-354818-        4921 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express EYFP with mitochondria-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4921)
  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 4921)
  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-at pHluorin
FEATURES             Location/Qualifiers
     source          1..4921
                     /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             768..1481
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(798..826)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1421..1441
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    1709..1830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1746..1765)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1800..1819
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1837..2292)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1974..1995)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2186..2205
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2319..2423
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2425..2782
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2425..2616
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2448..2468)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2633..2768
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2817..3611
                     /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             2817..3611
                     /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(2871..2890)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3481..3500
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3799..3818)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3843..3890
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4219..4807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4708..4727
                     /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 caacaccatg agtaaaggag
      781 aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc
      841 acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta
      901 aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc actacgttaa
      961 cttatggtgt tcaatgcttt tcaagatacc cagatcatat gaaacggcat gactttttca
     1021 agagtgccat gcccgaaggt tacgtacagg aaagaactat atttttcaaa gatgacggga
     1081 actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt
     1141 taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact
     1201 ataacgatca ccaggtgtac atcatggcag acaaacaaaa gaatggaatc aaagtgaact
     1261 tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa
     1321 atactccaat tggcgatggg cccgtccttt taccagacaa ccattacctg tttacaactt
     1381 ctactctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa
     1441 cagctgctgg gattacacat ggcatggatg aactatacaa aagatctctg actgactgag
     1501 atgcgcggat gtcgaacggc agttcggccg aataggccga cagccggccg tcggccgacg
     1561 acacggccag ggccggcggg gatccaccgg atctagataa ctgatcataa tcagccatac
     1621 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     1681 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     1741 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     1801 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     1861 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     1921 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     1981 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2041 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2101 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2161 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2221 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2281 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2341 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2401 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     2461 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2521 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2581 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     2641 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     2701 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     2761 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     2821 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     2881 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     2941 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3001 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3061 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3121 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3181 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3241 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3301 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3361 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3421 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3481 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3541 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3601 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3661 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3721 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3781 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     3841 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     3901 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     3961 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4021 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     4081 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4141 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4201 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4261 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4321 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4381 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4441 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4501 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4561 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4621 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4681 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4741 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4801 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4861 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     4921 t
//
