LOCUS       sequence-296409-        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"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             93..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             114..146
                     /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(150..158,159..173)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             183..890
                     /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(1480..1504)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1521..2255
                     /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     1658..1679
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1782..1803)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2043..2062)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2314..2332)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2328..2375
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2472..2927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2559..2578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2769..2790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2953..3057
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3058..3126,3127..3918)
                     /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(3276..3295)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4089..4677
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4578..4597
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4831..4848
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
       61 tttaagaagg agatatacat atgcggggtt ctcatcatca tcatcatcat ggtatggcta
      121 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatccga
      181 acatggtgag caagggcgag gagaccacaa tgggcgtaat caagcccgac atgaagatca
      241 agctgaagat ggagggcaac gtgaatggcc acgccttcgt gatcgagggc gagggcgagg
      301 gcaagcccta cgacggcacc aacaccatca acctggaggt gaaggaggga gcccccctgc
      361 ccttctccta cgacattctg accaccgcgt tcgcctacgg caacagggcc ttcaccaagt
      421 accccgacga catccccaac tacttcaagc agtccttccc cgagggctac tcttgggagc
      481 gcaccatgac cttcgaggac aagggcatcg tgaaggtgaa gtccgacatc tccatggagg
      541 aggactcctt catctacgag atacacctca agggcgagaa cttccccccc aacggccccg
      601 tgatgcagaa gaagaccacc ggctgggacg cctccaccga gaggatgtac gtgcgcgacg
      661 gcgtgctgaa gggcgacgtc aagcacaagc tgctgctgga gggcggcggc caccaccgcg
      721 ttgacttcaa gaccatctac agggccaaga aggcggtgaa gctgcccgac tatcactttg
      781 tggaccaccg catcgagatc ctgaaccacg acaaggacta caacaaggtg accgtttacg
      841 agagcgccgt ggcccgcaac tccaccgacg gcatggacga gctgtacaag agatctggta
      901 ccatggggat ggtcgaaatg aagcaggggg aagggacgta tgtgaaaagg tttgagcctg
      961 agcaaatttc catccccctt tcggctgcac ttttgatgaa aaagaaagat gtcgctgaac
     1021 tgctcgaagt ccgtaaaata cttgaaatcg gggcagtttc cagtgcggct caaaaacgca
     1081 cggaggatga tctcggacgc atgcaggaag cacttgagga tatgaagctg gcggacggga
     1141 acggcgaact tggcgaaaaa gccgacctcg cgtttcacct tgcgcttgcc ggcgcttcgc
     1201 agaacgatct gctgaaaggc ttgatgaatc atgtgtcatc tctcttaatt gagacgatga
     1261 gggagacgcg caaaatttgg ctgttctcga agaatacaac tgttaagcgc ctttatgagg
     1321 aacacgaagc catctatcag gctgtgaagg aaaaagacgg ccaaaaagcg gagcaagcga
     1381 tgcacaatca tctgacaaat gtggaaaatg tgctggcctc atattttgaa gaaacgaaac
     1441 aagccgacga gccggctatt cttaagaagg gcgaattcga cccagctttc ttgtacaaag
     1501 tggtgcttaa gagatctacc atggacggca gcgtgcagct cgccgaccac taccagcaga
     1561 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     1621 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1681 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggtggcagc ggtggcatgg
     1741 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1801 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1861 agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1921 tgaccaccct gggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     1981 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2041 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2101 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2161 tggagtacaa ctacaacagc cacaacgtct atatcaccgc cgacaagcag aagaacggca
     2221 tcaaggccaa cttcaagatc cgccacaaca tcgagtaaaa gcttgatccg gctgctaaca
     2281 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2341 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     2401 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2461 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2521 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2581 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2641 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2701 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2761 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2821 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2881 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2941 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3001 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3061 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3121 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3181 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3241 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3301 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3361 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3421 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3481 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3541 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3601 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3661 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3721 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3781 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3841 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3901 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3961 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4021 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4081 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4141 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4201 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4261 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4321 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4381 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4441 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4501 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4561 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4621 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4681 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4741 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4801 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4861 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg caggatctcg
     4921 atcccgcgaa at
//
