LOCUS       sequence-124615-        8369 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expresses pMito-eZinCh-2 Zn2+ FRET sensor in the mitochondria of 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8369)
  AUTHORS   Hessels AM, Chabosseau P, Bakker MH, Engelen W, Rutter GA, Taylor 
            KM, Merkx M
  TITLE     eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic
            and Intraorganelle Zn(2+) Imaging.
  JOURNAL   ACS Chem Biol. 2015 Sep 18;10(9):2126-34. doi: 
            10.1021/acschembio.5b00211. Epub 2015 Jul 20.
  PUBMED    26151333
REFERENCE   2  (bases 1 to 8369)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acschembio.5b00211"; journalName: "ACS Chem Biol"; date: 
            "2015-09-18- 18"; volume: "10"; issue: "9"; pages: "2126-34"
COMMENT     Sequence Label: pMito-eZinCh-2
FEATURES             Location/Qualifiers
     source          1..8369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             423..497
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      540..549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(555..557,558..560,561..1271)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSHLCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1473..1475,1476..1478,1479..2189)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSHLCKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5589..5606)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5760..6348)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5840..5859)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7161..7955
                     /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             7161..7955
                     /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(7215..7234)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7825..7844
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(8065..8086)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8277..8296
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttaattaann ntcccttcca gctctctgcc ccttttggat tgaagccaat atgataatga
       61 gggggtggag tttgtgacgt ggcgcggggc gtgggaacgg ggcgggtgac gtagtagtgt
      121 ggcggaagtg tgatgttgca agtgtggcgg aacacatgta agcgacggat gtggcaaaag
      181 tgacgttttt ggtgtgcgcc ggtgtacaca ggaagtgaca attttcgcgc ggttttaggc
      241 ggatgttgta gtaaatttgg gcgtaaccga gtaagatttg gccattttcg cgggaaaact
      301 gaataagagg aagtgaaatc tgaataattt tgtgttactc atagcgcgta annnggtacc
      361 gcggccgcct cgagcggcgg ctcccagtgc cgcgcgccaa gatccattcg ttgggggatc
      421 tcatgtccgt cctgacgccg ctgctgctgc ggggcttgac aggctcggcc cggcggctcc
      481 cagtgccgcg cgccaagatc cattcgttgc cgccggaggg agatctcaag agctcgagcg
      541 ccaccatggg ccatatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      601 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      661 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggt aagctgcccg
      721 tgccctggcc caccctcgtg accaccctga cctggggcgt gcagtgcttc gcccgctacc
      781 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      841 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      901 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      961 acatcctggg gcacaagctg gagtacaacg ccatcagcga caacgtctat atcaccgccg
     1021 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca
     1081 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1141 tgcccgacaa ccactacctg agcacccagt cccacctgtg caaagacccc aacgagaagc
     1201 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1261 agctgtacaa gtccggaggc ggcgagctca ttcgtggcgg atccggcgga agcggcggat
     1321 ccggcggtag cggcggatcc ggcggctccg gcggatccgg cggcagcggc ggatccggtg
     1381 gaagcggtgg atccggtggt agcggtggat ccggtggaag cggtggatcc ggtggtagcg
     1441 gtggatccgg gggtccgcgg ggctcggtac ccatggtgag caagggcgag gagctgttca
     1501 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1561 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1621 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctga
     1681 tgtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1741 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1801 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1861 acttcaagga ggacggcaac atcctggggc acaagcttga gtacaactac aacagccaca
     1921 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1981 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2041 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc cacctgtgca
     2101 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2161 tcactctcgg catggacgag ctgtacaagg tcgacaagga cgagctgggg ccctaaagcg
     2221 gccgctctag agatatcgaa ttcaagcttg tcgacagatc tnnntaaggg tgggaaagaa
     2281 tatataaggt gggggtctta tgtagttttg tatctgtttt gcagcagccg ccgccgccat
     2341 gagcaccaac tcgtttgatg gaagcattgt gagctcatat ttgacaacgc gcatgccccc
     2401 atgggccggg gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc ccgtcctgcc
     2461 cgcaaactct actaccttga cctacgagac cgtgtctgga acgccgttgg agactgcagc
     2521 ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg attgtgactg actttgcttt
     2581 cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg acaagttgac
     2641 ggctcttttg gcacaattgg attctttgac ccgggaactt aatgtcgttt ctcagcagct
     2701 gttggatctg cgccagcagg tttctgccct gaaggcttcc tcccctccca atgcggttta
     2761 aaacataaat aaaaaaccag actctgtttg gatttggatc aagcaagtgt cttgctgtct
     2821 ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt cgttgagggt
     2881 cctgtgtatt ttttccagga cgtggtaaag gtgactctgg atgttcagat acatgggcat
     2941 aagcccgtct ctggggtgga ggtagcacca ctgcagagct tcatgctgcg gggtggtgtt
     3001 gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt ctttcagtag
     3061 caagctgatt gccaggggca ggcccttggt gtaagtgttt acaaagcggt taagctggga
     3121 tgggtgcata cgtggggata tgagatgcat cttggactgt atttttaggt tggctatgtt
     3181 cccagccata tccctccggg gattcatgtt gtgcagaacc accagcacag tgtatccggt
     3241 gcacttggga aatttgtcat gtagcttaga aggaaatgcg tggaagaact tggagacgcc
     3301 cttgtgacct ccaagatttt ccatgcattc gtccataatg atggcaatgg gcccacgggc
     3361 ggcggcctgg gcgaagatat ttctgggatc actaacgtca tagttgtgtt ccaggatgag
     3421 atcgtcatag gccattttta caaagcgcgg gcggagggtg ccagactgcg gtataatggt
     3481 tccatccggc ccaggggcgt agttaccctc acagatttgc atttcccacg ctttgagttc
     3541 agatgggggg atcatgtcta cctgcggggc gatgaagaaa acggtttccg gggtagggga
     3601 gatcagctgg gaagaaagca ggttcctgag cagctgcgac ttaccgcagc cggtgggccc
     3661 gtaaatcaca cctattaccg ggtgcaactg gtagttaaga gagctgcagc tgccgtcatc
     3721 cctgagcagg ggggccactt cgttaagcat gtccctgact cgcatgtttt ccctgaccaa
     3781 atccgccaga aggcgctcgc cgcccagcga tagcagttct tgcaaggaag caaagttttt
     3841 caacggtttg agaccgtccg ccgtaggcat gcttttgagc gtttgaccaa gcagttccag
     3901 gcggtcccac agctcggtca cctgctctac ggcatctcga tccagcatat ctcctcgttt
     3961 cgcgggttgg ggcggctttc gctgtacggc agtagtcggt gctcgtccag acgggccagg
     4021 gtcatgtctt tccacgggcg cagggtcctc gtcagcgtag tctgggtcac ggtgaagggg
     4081 tgcgctccgg gctgcgcgct ggccagggtg cgcttgaggc tggtcctgct ggtgctgaag
     4141 cgctgccggt cttcgccctg cgcgtcggcc aggtagcatt tgaccatggt gtcatagtcc
     4201 agcccctccg cggcgtggcc cttggcgcgc agcttgccct tggaggaggc gccgcacgag
     4261 gggcagtgca gacttttgag ggcgtagagc ttgggcgcga gaaataccga ttccggggag
     4321 taggcatccg cgccgcaggc cccgcagacg gtctcgcatt ccacgagcca ggtgagctct
     4381 ggccgttcgg ggtcaaaaac caggtttccc ccatgctttt tgatgcgttt cttacctctg
     4441 gtttccatga gccggtgtcc acgctcggtg acgaaaaggc tgtccgtgtc cccgtataca
     4501 gactnnngtt taaacgaatt cnnntataaa atgcaaggtg ctgctcaaaa aatcaggcaa
     4561 agcctcgcgc aaaaaagaaa gcacatcgta gtcatgctca tgcagataaa ggcaggtaag
     4621 ctccggaacc accacagaaa aagacaccat ttttctctca aacatgtctg cgggtttctg
     4681 cataaacaca aaataaaata acaaaaaaac atttaaacat tagaagcctg tcttacaaca
     4741 ggaaaaacaa cccttataag cataagacgg actacggcca tgccggcgtg accgtaaaaa
     4801 aactggtcac cgtgattaaa aagcaccacc gacagctcct cggtcatgtc cggagtcata
     4861 atgtaagact cggtaaacac atcaggttga ttcatcggtc agtgctaaaa agcgaccgaa
     4921 atagcccggg ggaatacata cccgcaggcg tagagacaac attacagccc ccataggagg
     4981 tataacaaaa ttaataggag agaaaaacac ataaacacct gaaaaaccct cctgcctagg
     5041 caaaatagca ccctcccgct ccagaacaac atacagcgct tcacagcggc agcctaacag
     5101 tcagccttac cagtaaaaaa gaaaacctat taaaaaaaca ccactcgaca cggcaccagc
     5161 tcaatcagtc acagtgtaaa aaagggccaa gtgcagagcg agtatatata ggactaaaaa
     5221 atgacgtaac ggttaaagtc cacaaaaaac acccagaaaa ccgcacgcga acctacgccc
     5281 agaaacgaaa gccaaaaaac ccacaacttc ctcaaatcgt cacttccgtt ttcccacgtt
     5341 acgtaacttc ccattttaag aaaactacaa ttcccaacac atacaagtta ctccgcccta
     5401 aaacctacgt cacccgcccc gttcccacgc cccgcgccac gtcacaaact ccaccccctc
     5461 attatcatat tggcttcaat ccaaaataag gtatattatt gatgatnnnt taattaagga
     5521 tccnnncggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     5581 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5641 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5701 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5761 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5821 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5881 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5941 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6001 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6061 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6121 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6181 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6241 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6301 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6361 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6421 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6481 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6541 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6601 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6661 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6721 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6781 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgnnnn nnaaaaagga
     6841 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga
     6901 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
     6961 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg
     7021 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact
     7081 ggatggcttt ctcgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga
     7141 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     7201 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     7261 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     7321 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     7381 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     7441 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     7501 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     7561 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     7621 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     7681 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     7741 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     7801 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     7861 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     7921 tcgccttcta tcgccttctt gacgagttct tctgaatttt gttaaaattt ttgttaaatc
     7981 agctcatttt ttaaccaata ggccgaaatc ggcaacatcc cttataaatc aaaagaatag
     8041 accgcgatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     8101 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     8161 tcacccaaat caagtttttt gcggtcgagg tgccgtaaag ctctaaatcg gaaccctaaa
     8221 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     8281 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     8341 accaccacac ccgcgcgctt aatgcgccg
//
