LOCUS       sequence-161604-        4916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of redox sensitive GFP with a mitochondrial targeting 
            sequence in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4916)
  AUTHORS   Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, 
            Remington SJ
  TITLE     Investigating mitochondrial redox potential with redox-sensitive 
            green fluorescent protein indicators.
  JOURNAL   J Biol Chem. 2004 Mar 26;279(13):13044-53. Epub 2004 Jan 13.
  PUBMED    14722062
REFERENCE   2  (bases 1 to 4916)
  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"; journalName: "J Biol 
            Chem."; date: "2004-03-26"; volume: "279(13)"; pages: "13044-53. 
            Epub 2004 Jan 13"
COMMENT     Sequence Label: pEGFP-N1-mt-ro2GFP
FEATURES             Location/Qualifiers
     source          1..4916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        444..647
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     598..618
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      935..944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(941..943,944..946,947..1660)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(986..1007)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1247..1266)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1594..1615
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1783..1904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1820..1839)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1874..1893
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1911..2366)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2048..2069)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2260..2279
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2393..2497
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2499..2856
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2499..2690
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2522..2542)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2707..2842
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2891..3685
                     /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             2891..3685
                     /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(2945..2964)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3555..3574
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3873..3892)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3917..3964
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4293..4881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4782..4801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
       61 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
      121 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      181 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      241 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      301 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      361 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      421 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      481 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      541 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      601 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
      661 gtcagatccg ctagcgctac cggactcaga tctcgagctc gaattcgggc tgaaggagac
      721 ttgggggcac ccgcgtcgtg cctcctgggt tgtgaggagt cgccgctgcc gccactgcct
      781 gtgcttcatg aggaagatgc tcgccgccgt ctcccgcgtg ctgtctggcg cttctcagaa
      841 gccggcaagc agagtgctgg tagcatcccg taattttgca aatgatgcta catttctgca
      901 gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca agggcgagga
      961 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1021 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1081 catctccacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1141 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1201 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1261 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1321 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1381 ctgccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1441 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1501 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cctgctccgc
     1561 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1621 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     1681 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     1741 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     1801 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     1861 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     1921 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     1981 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2041 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2101 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2161 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2221 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2281 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2341 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     2401 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2461 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     2521 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2581 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2641 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2701 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2761 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     2821 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     2881 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2941 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     3001 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3061 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3121 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3181 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3241 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3301 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3361 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     3421 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3481 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3541 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3601 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3661 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     3721 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3781 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3841 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     3901 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     3961 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     4021 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     4081 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     4141 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4201 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4261 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4321 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4381 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4441 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4501 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4561 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4621 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4681 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4741 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4801 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4861 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggc
//
