LOCUS       sequence-476346-        6845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mitochondria-targeted red fluorescent 
            pyruvate control biosensor dApplePy1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6845)
  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 6845)
  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-dApplePy1
FEATURES             Location/Qualifiers
     source          1..6845
                     /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(2311..2328)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2317..2541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2587..3015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2674..2693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2884..2905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3024..3044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3029..3358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3209..3344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3271..3290
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3425..4219
                     /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(3479..3498)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4089..4108
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4393..4514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4430..4449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4484..4503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4563..4579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4563..4579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4576..4598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4587..4603
                     /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(4611..4617,4618..4635,4636..4641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4656..4677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4794..4811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4965..5553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5045..5064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5724..6515,6516..6584))
                     /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     6347..6366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6585..6689)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6764..6783)
                     /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 ctgctagaac 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 tgctgagata tatggagcct atgctcgcac agaatatccg
     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 ggttggggac gacgagagat
     2101 gctgccgctc gtggcctccc atcgcactcg catatttgaa gctattatgg caggaaaacc
     2161 ggaggaagcc agggaagcgt cacatagaca cttggccttt attaaggaaa tcctgctgga
     2221 ccggtcaagg ggggaatcac gccgcgaact ctcattgaga aggctggagc aaagagagaa
     2281 ttgaaagctt aagtttaaac cgctgatcag cctcgactgt gccttctagt tgccagccat
     2341 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2401 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2461 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2521 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     2581 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2641 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2701 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2761 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2821 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2881 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2941 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3001 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3121 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3181 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3241 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3301 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3361 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3421 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3481 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3541 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3601 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3661 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3721 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3781 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3841 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3901 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3961 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4021 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4081 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4141 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4201 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4261 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4321 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4381 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4441 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4501 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     4561 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4621 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4681 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4741 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4801 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4861 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4921 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4981 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5041 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5101 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5161 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5221 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5281 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5341 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5401 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5461 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5521 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5581 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5641 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5701 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5761 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5821 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5881 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5941 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6001 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6061 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6121 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6181 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6241 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6301 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6361 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6421 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6481 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6541 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6601 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6661 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6721 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     6781 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     6841 cgagc
//
