LOCUS       sequence-296408-        8295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET-Based Genetically Encoded Nanosensor for Lactate/Pyruvate 
            Ratio.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8295)
  AUTHORS   Galaz A, Cortes-Molina F, Arce-Molina R, Romero-Gomez I, Mardones 
            GA, Barros LF, San Martin A
  TITLE     Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded 
            FRET Indicator.
  JOURNAL   Anal Chem. 2020 Jun 29. doi: 10.1021/acs.analchem.0c01741.
  PUBMED    32600029
REFERENCE   2  (bases 1 to 8295)
  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: "Anal Chem. 
            2020 Jun 29. doi: 10.1021/acs.analchem.0c01741."
COMMENT     Sequence Label: Mito-Lapronic/pGW1
FEATURES             Location/Qualifiers
     source          1..8295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          720..1546
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             1752..1826
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1848..1922
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1944..2018
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2040..2114
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2145..2852
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     protein_bind    complement(3442..3466)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3483..4217
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     3620..3641
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3744..3765)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4005..4024)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    4351..4485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4388..4407)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4442..4461
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4508..4524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4508..4524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4521..4543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4532..4548
                     /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(4556..4562,4563..4580,4581..4586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4738..4755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4909..5497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4989..5008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5668..6459,6460..6528))
                     /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     6291..6310
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6585..7098)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6780..6801)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6992..7011
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7270..7288
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7326..7348)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7448..7467
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7699..7721
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7713..7730
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7714..7730
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcttatgca tgctatactg
      781 tttttggctt ggggtctata cacccccgct tcctcatgtt ataggtgatg gtatagctta
      841 gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc
      901 cattactaat ccataacatg gctctttgcc acaactctct ttattggcta tatgccaata
      961 cactgtcctt cagagactga cacggactct gtatttttac aggatggggt ctcatttatt
     1021 atttacaaat tcacatatac aacaccaccg tccccagtgc ccgcagtttt tattaaacat
     1081 aacgtgggat ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta
     1141 gcggcggagc ttctacatcc gagccctgct cccatgcctc cagcgactca tggtcgctcg
     1201 gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacgatg cccaccacca
     1261 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag ctcggggagc
     1321 gggcttgcac cgctgacgca tttggaagac ttaaggcagc ggcagaagaa gatgcaggca
     1381 gctgagttgt tgtgttctga taagagtcag aggtaactcc cgttgcggtg ctgttaacgg
     1441 tggagggcag tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata
     1501 gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtccttgac
     1561 acgaagcttc ccggccacag caatcgatcc atgacacaac cagtgacaag cagtccccca
     1621 gtgaaaccgc atccgcatcc gagtccgata ccgataaaga ttctgaatcg gagtgagtgc
     1681 cgcgtccgag agcgttccct gtccacgtcc accatcggcg gagcaggtgt gcctgaggcc
     1741 tctagaccac catgagcgtg ctgacaccat tgttgctcag aggcctcact ggttcagcca
     1801 ggcggctgcc agtcccaaga gctaagatcc atagtctggg agatccaatg tcagttctca
     1861 ctcccctttt gcttagggga ctgaccggaa gcgctcggcg gttgcctgtc ccacgcgcca
     1921 agattcactc cctgggcgac cctatgagtg tccttactcc tcttttgctg agaggcttga
     1981 cagggagcgc tcggaggctc ccagtgcctc gcgcaaagat ccattctctc ggagatccca
     2041 tgtccgtcct cactcctctg ctgctgcgcg ggcttacagg ttccgctcgc cgcctgcctg
     2101 ttcctcgggc aaagatacac agtctgggcg atcctcaccg gcgcatggtg agcaagggcg
     2161 aggagaccac aatgggcgta atcaagcccg acatgaagat caagctgaag atggagggca
     2221 acgtgaatgg ccacgccttc gtgatcgagg gcgagggcga gggcaagccc tacgacggca
     2281 ccaacaccat caacctggag gtgaaggagg gagcccccct gcccttctcc tacgacattc
     2341 tgaccaccgc gttcgcctac ggcaacaggg ccttcaccaa gtaccccgac gacatcccca
     2401 actacttcaa gcagtccttc cccgagggct actcttggga gcgcaccatg accttcgagg
     2461 acaagggcat cgtgaaggtg aagtccgaca tctccatgga ggaggactcc ttcatctacg
     2521 agatacacct caagggcgag aacttccccc ccaacggccc cgtgatgcag aagaagacca
     2581 ccggctggga cgcctccacc gagaggatgt acgtgcgcga cggcgtgctg aagggcgacg
     2641 tcaagcacaa gctgctgctg gagggcggcg gccaccaccg cgttgacttc aagaccatct
     2701 acagggccaa gaaggcggtg aagctgcccg actatcactt tgtggaccac cgcatcgaga
     2761 tcctgaacca cgacaaggac tacaacaagg tgaccgttta cgagagcgcc gtggcccgca
     2821 actccaccga cggcatggac gagctgtaca agagatctgg taccatgggg atggtcgaaa
     2881 tgaagcaggg ggaagggacg tatgtgaaaa ggtttgagcc tgagcaaatt tccatccccc
     2941 tttcggctgc acttttgatg aaaaagaaag atgtcgctga actgctcgaa gtccgtaaaa
     3001 tacttgaaat cggggcagtt tccagtgcgg ctcaaaaacg cacggaggat gatctcggac
     3061 gcatgcagga agcacttgag gatatgaagc tggcggacgg gaacggcgaa cttggcgaaa
     3121 aagccgacct cgcgtttcac cttgcgcttg ccggcgcttc gcagaacgat ctgctgaaag
     3181 gcttgatgaa tcatgtgtca tctctcttaa ttgagacgat gagggagacg cgcaaaattt
     3241 ggctgttctc gaagaataca actgttaagc gcctttatga ggaacacgaa gccatctatc
     3301 aggctgtgaa ggaaaaagac ggccaaaaag cggagcaagc gatgcacaat catctgacaa
     3361 atgtggaaaa tgtgctggcc tcatattttg aagaaacgaa acaagccgac gagccggcta
     3421 ttcttaagaa gggcgaattc gacccagctt tcttgtacaa agtggtgctt aagagatcta
     3481 ccatggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3541 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     3601 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3661 tcggcatgga cgagctgtac aagggtggca gcggtggcat ggtgagcaag ggcgaggagc
     3721 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3781 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga
     3841 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg
     3901 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3961 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     4021 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     4081 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     4141 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     4201 tccgccacaa catcgagtaa gctagctgag tcgacgaatt cattgatcat aatcagccat
     4261 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4321 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4381 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4441 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgtac cgagctcgcg
     4501 taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4561 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactacatt
     4621 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4681 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5341 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5581 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5641 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5701 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5761 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5821 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5881 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5941 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6001 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6061 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6121 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6181 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6241 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6301 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6361 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6421 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6481 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6541 tcaataagcg gccgcggcca tgccggccac tagtctcgag ggccaccccg gttgataatc
     6601 agaaaagccc caaaaacagg aagattgtat aagcaaatat ttaaattgta agcgttaata
     6661 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     6721 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     6781 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     6841 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     6901 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6961 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     7021 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     7081 cgccgctaca gggcgcgtgg ggatccctcg agttattatt gaagcattta tcagggttat
     7141 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7201 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7261 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     7321 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     7381 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     7441 aactatgcgg catcagagca gattgtactg agagtgcacc atatgaagac gtcgcctcct
     7501 cactacttct ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa
     7561 attagtcagg gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     7621 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     7681 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gcccgtgtca
     7741 aggacggtga ctgcagtgaa taataaaatg tgtgtttgtc cgaaatacgc gttttgagat
     7801 ttctgtcgcc gactaaattc atgtcgcgcg atagtggtgt ttatcgccga tagagatggc
     7861 gatattggaa aaatcgatat ttgaaaatat ggcatattga aaatgtcgcc gatgtgagtt
     7921 tctgtgtaac tgatatcgcc atttttccaa aagtgatttt tgggcatacg cgatatctgg
     7981 cgatagcgct tatatcgttt acgggggatg gcgatagacg actttggtga cttgggcgat
     8041 tctgtgtgtc gcaaatatcg cagtttcgat ataggtgaca gacgatatga ggctatatcg
     8101 ccgatagagg cgacatcaag ctggcacatg gccaatgcat atcgatctat acattgaatc
     8161 aatattggcc attagccata ttattcattg gttatatagc ataaatcaat attggctatt
     8221 ggccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa
     8281 cattaccgcc atgtt
//
