LOCUS       sequence-210136-        7355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mitochondria targetting gTEMP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7355)
  AUTHORS   Nakano M, Arai Y, Kotera I, Okabe K, Kamei Y, Nagai T
  TITLE     Genetically encoded ratiometric fluorescent thermometer with wide 
            range and rapid response.
  JOURNAL   PLoS One. 2017 Feb 17;12(2):e0172344. doi: 
            10.1371/journal.pone.0172344. eCollection 2017.
  PUBMED    28212432
REFERENCE   2  (bases 1 to 7355)
  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: "PLoS One.";
            date: "2017-02-17"; volume: "12(2):e0172344. doi"; pages: " 
            10.1371/journal.pone.0172344. eCollection 2017"
COMMENT     Sequence Label: mito-gTEMP_pcDNA3
FEATURES             Location/Qualifiers
     source          1..7355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        129..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        509..712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     663..683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     757..776
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        757..775
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             813..887
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             909..983
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1485..1736
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKAHFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSVQSKLSKDPNEKRDHMVLLESVTAAGITLGMDELYK"
     CDS             1755..1808
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1815..1889
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1911..1985
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(2022..2024,2025..2027,2028..2738)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(2802..2820)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2803..2820)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2840..2857)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2846..3070
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3116..3544
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3203..3222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3413..3434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3553..3573)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3558..3887
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3738..3873
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3800..3819
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3954..4748
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4008..4027)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4618..4637
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4922..5043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4959..4978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5013..5032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5092..5108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5092..5108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5105..5127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5116..5132
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5140..5146,5147..5164,5165..5170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5185..5206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5323..5340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5494..6082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5574..5593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6253..7044,7045..7113))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6876..6895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7114..7218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7293..7312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
       61 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
      721 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      781 ccaagcttgg taccgagctc ggatccgcca ccatgtctgt tctgactcct ctgctgctcc
      841 ggggtctcac aggttccgca agaagactcc ccgtgcctag ggccaaaatt cattcactgg
      901 gggaccccat gagcgtgctc accccactcc tgctgcgggg gctgaccggc agcgctaggc
      961 ggctgccagt ccccagggcc aagatccaca gtctcggcga tcccaagcgc ggccgcatca
     1021 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1081 gcgacgtaaa cggccacagg ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1141 gcaagctgac cctgaagctc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1201 tcgtgaccac cctgcagttc ggcgtgctgt gcttcgcccg ctaccccgac cacatgaagc
     1261 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1321 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1381 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1441 agctggagta caacgggata agctcaaacg tatatatcac cgccgacaag cagaagaacg
     1501 gcatcaaggc ccacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg
     1561 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1621 acctgagcgt ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1681 tgctggagtc cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagcgca
     1741 tgcatggcag cggcgagggc agaggcagcc tgctgacctg cggcgacgtg gaggagaacc
     1801 ccggccccga gctcatgtct gttctgactc ctctgctgct ccggggtctc acaggttccg
     1861 caagaagact ccccgtgcct agggccaaaa ttcattcact gggggacccc atgagcgtgc
     1921 tcaccccact cctgctgcgg gggctgaccg gcagcgctag gcggctgcca gtccccaggg
     1981 ccaagatcca cagtctcggc gatcccaagc gcggccgcat catggtgagc aagggcgagg
     2041 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2101 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2161 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttctcct
     2221 acggcgtgat ggtgttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2281 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2341 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2401 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca
     2461 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gccaacttca
     2521 agatccgcca caacatcgag gacggcggcg tgcagctcgc cgaccactac cagcagaaca
     2581 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc atccagtcca
     2641 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2701 ccgccgggat cactctcggc atggacgagc tgtacaagta agaattctgc agatatccat
     2761 cacactggcg gccgctcgag catgcatcta gagggcccta ttctatagtg tcacctaaat
     2821 gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2881 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2941 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3001 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3061 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3121 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3181 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3241 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3301 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3361 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3421 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3481 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3541 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3601 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3661 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3721 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3781 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3841 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3901 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3961 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4021 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4081 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4141 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4201 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4261 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4321 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4381 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4441 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4501 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4561 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4621 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4681 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4741 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4801 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4861 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4921 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4981 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5041 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5101 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5161 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6121 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6181 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6301 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6361 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6421 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6481 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6541 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6601 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6661 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6721 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6781 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6841 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6901 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6961 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7021 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7081 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7141 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7201 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7261 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7321 agccagtatc tgctccctgc ttgtgtgttg gaggt
//
