LOCUS       sequence-476340-        6842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mitochondria-targeted lowest affinity red 
            fluorescent pyruvate biosensor ApplePy1Lowest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6842)
  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 6842)
  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-ApplePy1Lowest
FEATURES             Location/Qualifiers
     source          1..6842
                     /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     complement(2308..2325)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2314..2538
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2584..3012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2671..2690)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2881..2902
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3021..3041)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3026..3355
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3206..3341
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3268..3287
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3422..4216
                     /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(3476..3495)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4086..4105
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4390..4511
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4427..4446)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4481..4500
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4560..4576)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4560..4576)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4573..4595)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4584..4600
                     /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(4608..4614,4615..4632,4633..4638))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4653..4674
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4791..4808)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4962..5550)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5042..5061)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5721..6512,6513..6581))
                     /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     6344..6363
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6582..6686)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6761..6780)
                     /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 ctccgatccg ctggtagaac tgctttccga tcacccggaa tcccaatatg acttgctcga
     1081 aacgcgccat gcgctggaag ggatcgcagc atattacgct gccttgtgca gctcagatga
     1141 agagaaagag aggatcagag agttgcatca tgcgattgag cttgcccaac aaagtggtga
     1201 tctcgatgcc gaaagtaacg ctgtacttca gtatcaaatc gcggtgaccg aggcagcgca
     1261 caatgtggtc cttttgcact tgttgagata tatggagcct atgctcgcac agaatgtccg
     1321 acataacttt gagctgctgt actctggtac ggtggtttcc gagcggatgt tccccgagga
     1381 cggcgccctg aagagcgagg tcaagaaggg gctgaggctg aaggacggcg gccactacgc
     1441 cgtggaggtc aagaccacct acaaggccaa gaagcccgtg cccctgcccg gcgcctacat
     1501 cgtcgacatc atgttggaca tcgtgtccca caacgaggac tacactatcg tggaacagtt
     1561 cgaacgcgcc gagggccgcc accccaccgg cggcctggac gagctgtaca agggaggtac
     1621 aggcgggagt ctggtaagca agggcgagga ggatgacatg gccatcgtca aggagttcat
     1681 gcgcttcaag gtgcacatgg agggctccgt taacggccac gagttcgaga tcgaaggcga
     1741 gggcgagggc cgcccctacg aggcctttca gactgctaag ctgaaggtga ccaagggtgg
     1801 ccccctgccc ttcgcctggg acatcttatc ccctcagttc atgtacggct ccaaggccta
     1861 cattaagcac ccagccgata tccccgacta cttcaagctg tccttccccg agggcttcag
     1921 ctgggagcgc gtgatgtact tcgaggacgg cggcattatt cacgttaacc aggactcctc
     1981 cctgcaggac ggcgtattta tctacaaggt gaagctgcgc ggcaccaact tcccccccga
     2041 cggccccgta atgcagatga agaccatggg ctgggagtcg ggttggcgac gagagatgct
     2101 gccgctcgtg gcctcccatc gcactcgcat atttgaagct attatggcag gaaaaccgga
     2161 ggaagccagg gaagcgtcac atagacactt ggcctttatt aaggaaatcc tgctggaccg
     2221 gtcaaggggg gaatcacgcc gcgaactctc attgagaagg ctggagcaaa gagagaattg
     2281 aaagcttaag tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2341 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2401 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2461 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2521 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2581 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2641 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2701 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2761 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2821 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2881 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2941 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3001 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3061 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3121 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3181 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3241 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3301 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3361 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3421 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3481 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3541 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3601 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3661 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3721 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3781 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3841 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3901 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3961 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4021 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4081 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4141 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4201 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4261 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4321 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4381 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4441 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4501 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4561 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4621 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4681 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4741 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4801 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4861 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4921 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4981 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5041 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5101 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5161 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5221 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5281 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5341 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5401 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5461 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5521 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5581 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5641 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5701 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5761 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5821 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5881 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5941 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6001 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6061 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6121 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6181 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6241 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6301 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6361 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6421 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6481 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6541 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6601 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6661 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     6721 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     6781 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     6841 gc
//
