LOCUS       sequence-476348-        6836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mitochondria-targeted green fluorescent 
            pyruvate control biosensor dGreenPy1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6836)
  AUTHORS   Imai S, Hario S, Suerte C, Yamaguchi I, Sakoi K, Terai T, 
            Takahashi-Yamashiro K, Campbell RE
  TITLE     High-performance genetically-encoded green and red fluorescent 
            biosensors for pyruvate
  JOURNAL   bioRxiv 2025.04.17.649293
REFERENCE   2  (bases 1 to 6836)
  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: "bioRxiv 
            2025.04.17.649293"
COMMENT     Sequence Label: pcDNA-mito-dGreenPy1
FEATURES             Location/Qualifiers
     source          1..6836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     738..757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             803..877
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             899..973
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     1555..1576
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1682..1703)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1943..1962)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2302..2319)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2308..2532
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2578..3006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2665..2684)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2875..2896
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3015..3035)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3020..3349
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3200..3335
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3262..3281
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3416..4210
                     /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(3470..3489)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4080..4099
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4384..4505
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4421..4440)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4475..4494
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4554..4570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4554..4570)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4567..4589)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4578..4594
                     /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(4602..4608,4609..4626,4627..4632))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4647..4668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4785..4802)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4956..5544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5036..5055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5715..6506,6507..6575))
                     /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     6338..6357
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6576..6680)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6755..6774)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      721 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta
      781 aacgggccct ctagactcga gcatgtctgt tctgactcct ctgctgctcc ggggtctcac
      841 aggttccgca agaagactcc ccgtgcctag ggccaaaatt cattcactgg gggaccccat
      901 gagcgtgctc accccactcc tgctgcgggg gctgaccggc agcgctaggc ggctgccagt
      961 ccccagggcc aagatccaca gtctcggcga tcccaaggat ccaaccatgg tgcaatcttt
     1021 ttccgatccg ctggtagaac tgcttccaga tcacccggaa tcccaatatg acttgctcga
     1081 agcgcgccat gcgctggaag ggatcgcagc atattacgct gccttgcgca gcacagatga
     1141 agataaagag aggatcagag agttgcatca tgcgattggg cttgcccaac aaagtggtga
     1201 tctcgatgcc gaaagtaacg ctgtacttca gtatcaaacc gcggtgaccg aggcagcgca
     1261 caatgtggtc cttttgcact tgttgagatg tatggagcct atgctcgcac agaatatccg
     1321 acaaaacttt gagctgctgt accgtcataa cgtctatgtc atggccgaca agcagaggaa
     1381 cggcatcaag gcgaacttca agatccgcca caacatcgag ggcggcggcg tgcagctcgc
     1441 ctaccaccac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1501 ctacctgtgc acccagtcca aactttcgaa agaccccaac gagaagcgcg atcacatggt
     1561 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacatggg
     1621 cggtaccgga gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1681 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggtga
     1741 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1801 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcggccgcta
     1861 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacatcca
     1921 ggagcgcacc atcttcttcg aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1981 cgagggcgac accctggtga gccgcatcga gctgaagggc atcgacttca aggaggacgg
     2041 caacatcctg gggcataagc tggagtacaa ctcgtggggt cgacgagaga tgctgccgct
     2101 cgtgtcttcc catcgcactc gcatatttga agctattatg gcaggaaaac cggaggaagc
     2161 cagggaagcg tcccataaac acttggcctt tattgaggaa atcctgctgg accggtcaag
     2221 ggaggaatca cgccgcgaac gctcattgag acggctggag caaagaaaga attgaaagct
     2281 taagtttaaa ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2341 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2401 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2461 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2521 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2581 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2641 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2701 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2761 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2821 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2881 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2941 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3001 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3061 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3121 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3181 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3241 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3301 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3361 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3421 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3481 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3541 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3601 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3661 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3721 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3781 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3841 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3901 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     3961 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4021 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4081 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4141 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4201 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4261 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4321 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4381 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4441 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4501 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4561 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4621 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4681 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4741 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4801 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4861 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4921 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4981 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5041 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5101 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5161 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5221 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5281 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5341 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5401 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5461 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5521 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5581 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5641 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5701 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5761 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5821 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5881 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5941 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6001 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6061 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6121 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6181 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6241 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6301 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6361 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6421 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6481 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6541 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6601 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6661 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     6721 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6781 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagc
//
