LOCUS       sequence-476970-        6833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mitochondria-targeted lowest affinity green 
            fluorescent pyruvate biosensor GreenPy1Lowest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6833)
  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 6833)
  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-GreenPy1Lowest
FEATURES             Location/Qualifiers
     source          1..6833
                     /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(2299..2316)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2305..2529
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2575..3003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2662..2681)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2872..2893
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3012..3032)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3017..3346
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3197..3332
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3259..3278
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3413..4207
                     /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(3467..3486)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4077..4096
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4381..4502
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4418..4437)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4472..4491
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4551..4567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4551..4567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4564..4586)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4575..4591
                     /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(4599..4605,4606..4623,4624..4629))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4644..4665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4782..4799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4953..5541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5033..5052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5712..6503,6504..6572))
                     /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     6335..6354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6573..6677)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6752..6771)
                     /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 agaatgtccg
     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 ctcgtggcga cgagagatgc tgccgctcgt
     2101 gtcttcccat cgcactcgca tatttgaagc tattatggca ggaaaaccgg aggaagccag
     2161 ggaagcgtcc cataaacact tggcctttat tgaggaaatc ctgctggacc ggtcaaggga
     2221 ggaatcacgc cgcgaacgct cattgagacg gctggagcaa agaaagaatt gaaagcttaa
     2281 gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2341 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2401 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2461 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2521 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2581 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2641 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2701 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2761 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2821 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2881 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2941 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3001 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3061 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3121 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3181 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3241 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     3301 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3361 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3421 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3481 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3541 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3601 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3661 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3721 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3781 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3841 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3901 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3961 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4021 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4081 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4141 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4201 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4261 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4321 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4381 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4441 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4501 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4561 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4621 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4681 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4741 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4801 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4861 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4921 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4981 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5041 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5101 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5161 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5221 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5281 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5341 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5401 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5461 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5521 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5581 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5641 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5701 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5761 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5821 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5881 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5941 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6001 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6061 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6121 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6181 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6241 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6301 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6361 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6421 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6481 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6541 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6601 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6661 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     6721 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     6781 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agc
//
