LOCUS       sequence-296407-        7498 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 7498)
  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 7498)
  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: Lapronic/pcDNA3.1(-)
FEATURES             Location/Qualifiers
     source          1..7498
                     /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     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             753..1460
                     /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(2050..2074)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2091..2825
                     /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     2228..2249
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2352..2373)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2613..2632)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2855..2872)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2861..3085
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3131..3559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3218..3237)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3428..3449
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3568..3588)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3573..3902
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3753..3888
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3815..3834
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3969..4763
                     /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(4023..4042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4633..4652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4937..5058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4974..4993)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5028..5047
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5107..5123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5107..5123)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5120..5142)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5131..5147
                     /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(5155..5161,5162..5179,5180..5185))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5200..5221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5338..5355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5509..6097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5589..5608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6268..7059,7060..7128))
                     /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     6891..6910
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7129..7233)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7308..7327)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
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 agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacgggccc tctagactcg agcggccgcc actgtgctgg atatctgcag
      721 aattccacca cactggacta gtggatccga acatggtgag caagggcgag gagaccacaa
      781 tgggcgtaat caagcccgac atgaagatca agctgaagat ggagggcaac gtgaatggcc
      841 acgccttcgt gatcgagggc gagggcgagg gcaagcccta cgacggcacc aacaccatca
      901 acctggaggt gaaggaggga gcccccctgc ccttctccta cgacattctg accaccgcgt
      961 tcgcctacgg caacagggcc ttcaccaagt accccgacga catccccaac tacttcaagc
     1021 agtccttccc cgagggctac tcttgggagc gcaccatgac cttcgaggac aagggcatcg
     1081 tgaaggtgaa gtccgacatc tccatggagg aggactcctt catctacgag atacacctca
     1141 agggcgagaa cttccccccc aacggccccg tgatgcagaa gaagaccacc ggctgggacg
     1201 cctccaccga gaggatgtac gtgcgcgacg gcgtgctgaa gggcgacgtc aagcacaagc
     1261 tgctgctgga gggcggcggc caccaccgcg ttgacttcaa gaccatctac agggccaaga
     1321 aggcggtgaa gctgcccgac tatcactttg tggaccaccg catcgagatc ctgaaccacg
     1381 acaaggacta caacaaggtg accgtttacg agagcgccgt ggcccgcaac tccaccgacg
     1441 gcatggacga gctgtacaag agatctggta ccatggggat ggtcgaaatg aagcaggggg
     1501 aagggacgta tgtgaaaagg tttgagcctg agcaaatttc catccccctt tcggctgcac
     1561 ttttgatgaa aaagaaagat gtcgctgaac tgctcgaagt ccgtaaaata cttgaaatcg
     1621 gggcagtttc cagtgcggct caaaaacgca cggaggatga tctcggacgc atgcaggaag
     1681 cacttgagga tatgaagctg gcggacggga acggcgaact tggcgaaaaa gccgacctcg
     1741 cgtttcacct tgcgcttgcc ggcgcttcgc agaacgatct gctgaaaggc ttgatgaatc
     1801 atgtgtcatc tctcttaatt gagacgatga gggagacgcg caaaatttgg ctgttctcga
     1861 agaatacaac tgttaagcgc ctttatgagg aacacgaagc catctatcag gctgtgaagg
     1921 aaaaagacgg ccaaaaagcg gagcaagcga tgcacaatca tctgacaaat gtggaaaatg
     1981 tgctggcctc atattttgaa gaaacgaaac aagccgacga gccggctatt cttaagaagg
     2041 gcgaattcga cccagctttc ttgtacaaag tggtgcttaa gagatctacc atggacggca
     2101 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2161 tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc
     2221 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2281 agctgtacaa gggtggcagc ggtggcatgg tgagcaaggg cgaggagctg ttcaccgggg
     2341 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2401 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc tgcaccaccg
     2461 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc ctgcagtgct
     2521 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2581 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2641 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2701 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2761 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca
     2821 tcgagtaaaa gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag
     2881 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2941 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3001 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3061 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3121 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3181 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3241 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3301 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3361 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3421 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3481 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3541 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3601 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3661 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3721 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3781 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3841 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3901 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3961 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4021 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4081 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4141 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4201 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4261 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4321 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4381 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4441 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4501 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4561 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4621 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4681 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4741 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4801 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4921 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4981 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5041 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5101 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5161 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5221 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5281 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5341 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5401 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5461 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5521 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5581 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5641 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5701 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5761 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5821 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5881 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5941 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6001 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6061 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6121 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6181 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6241 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6301 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6361 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6421 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6481 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6541 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6601 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6661 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6721 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6781 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6841 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6901 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6961 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7021 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7081 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7141 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7201 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7261 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     7321 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     7381 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     7441 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
