LOCUS       sequence-273406-        4932 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 4932)
  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 4932)
  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/pRSETB
FEATURES             Location/Qualifiers
     source          1..4932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             31..765
                     /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     168..189
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(292..313)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(553..572)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(824..842)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      838..885
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      982..1437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1069..1088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1279..1300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1463..1567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1568..1636,1637..2428)
                     /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     complement(1786..1805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2599..3187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3088..3107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3341..3358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3443..3462
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3443..3461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3493..3515
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3535..3552
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3556..3588
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(3592..3600,3601..3615)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             3625..4332
                     /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(join(4922..4932,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggtgcttaa gagatctacc atggacggca gcgtgcagct cgccgaccac
       61 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      121 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
      181 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggtggcagc
      241 ggtggcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      301 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      361 acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg
      421 cccaccctcg tgaccaccct gggctacggc ctgcagtgct tcgcccgcta ccccgaccac
      481 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      541 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      601 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      661 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcaccgc cgacaagcag
      721 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgagtaaaa gcttgatccg
      781 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
      841 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
      901 atatccggat ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
      961 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1021 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1081 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1141 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1201 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1261 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1321 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1381 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1441 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1501 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1561 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1621 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1681 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1741 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1801 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1861 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     1921 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1981 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2041 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2101 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2161 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2221 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2281 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2341 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2401 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2461 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2521 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2581 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2641 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2701 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     2761 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2821 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2881 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2941 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3001 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3061 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3121 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3181 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3241 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3301 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3361 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     3421 caggatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc
     3481 tctagaaata attttgttta actttaagaa ggagatatac atatgcgggg ttctcatcat
     3541 catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac
     3601 gacgatgacg ataaggatcc gaacatggtg agcaagggcg aggagaccac aatgggcgta
     3661 atcaagcccg acatgaagat caagctgaag atggagggca acgtgaatgg ccacgccttc
     3721 gtgatcgagg gcgagggcga gggcaagccc tacgacggca ccaacaccat caacctggag
     3781 gtgaaggagg gagcccccct gcccttctcc tacgacattc tgaccaccgc gttcgcctac
     3841 ggcaacaggg ccttcaccaa gtaccccgac gacatcccca actacttcaa gcagtccttc
     3901 cccgagggct actcttggga gcgcaccatg accttcgagg acaagggcat cgtgaaggtg
     3961 aagtccgaca tctccatgga ggaggactcc ttcatctacg agatacacct caagggcgag
     4021 aacttccccc ccaacggccc cgtgatgcag aagaagacca ccggctggga cgcctccacc
     4081 gagaggatgt acgtgcgcga cggcgtgctg aagggcgacg tcaagcacaa gctgctgctg
     4141 gagggcggcg gccaccaccg cgttgacttc aagaccatct acagggccaa gaaggcggtg
     4201 aagctgcccg actatcactt tgtggaccac cgcatcgaga tcctgaacca cgacaaggac
     4261 tacaacaagg tgaccgttta cgagagcgcc gtggcccgca actccaccga cggcatggac
     4321 gagctgtaca agagatctgg taccatgggg atggtcgaaa tgaagcaggg ggaagggacg
     4381 tatgtgaaaa ggtttgagcc tgagcaaatt tccatccccc tttcggctgc acttttgatg
     4441 aaaaagaaag atgtcgctga actgctcgaa gtccgtaaaa tacttgaaat cggggcagtt
     4501 tccagtgcgg ctcaaaaacg cacggaggat gatctcggac gcatgcagga agcacttgag
     4561 gatatgaagc tggcggacgg gaacggcgaa cttggcgaaa aagccgacct cgcgtttcac
     4621 cttgcgcttg ccggcgcttc gcagaacgat ctgctgaaag gcttgatgaa tcatgtgtca
     4681 tctctcttaa ttgagacgat gagggagacg cgcaaaattt ggctgttctc gaagaataca
     4741 actgttaagc gcctttatga ggaacacgaa gccatctatc aggctgtgaa ggaaaaagac
     4801 ggccaaaaag cggagcaagc gatgcacaat catctgacaa atgtggaaaa tgtgctggcc
     4861 tcatattttg aagaaacgaa acaagccgac gagccggcta ttcttaagaa gggcgaattc
     4921 gacccagctt tc
//
