LOCUS       sequence-441743-        6670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Biosensor for extracellular L-lactate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6670)
  AUTHORS   Kamijo Y, Machler P, Ness N, Vu CQ, Kusakizako T, Mannuthodikayil J,
            Ku Z, Boisvert M, Grebenik E, Miyazaki I, Hashizume R, Sato H, Liu 
            R, Hori Y, Tomita T, Katayama T, Furube A, Caraveo G, Paquet ME, 
            Drobizhev M, Nureki O, Arai S, Brancaccio M, Campbell RE, Kleinfeld 
            D, Nasu Y
  TITLE     A red fluorescent genetically encoded biosensor for in vivo imaging 
            of extracellular L-lactate dynamics.
  JOURNAL   Nat Commun. 2025 Oct 29;16(1):9531. doi: 10.1038/s41467-025-64484-x.
  PUBMED    41162396
REFERENCE   2  (bases 1 to 6670)
  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: "Nat 
            Commun."; date: "2025-10-29"; volume: "16(1):9531. doi"; pages: " 
            10.1038/s41467-025-64484-x"
COMMENT     Sequence Label: pAAV-hSyn-IgK-R-eLACCO2.1-COBRA
FEATURES             Location/Qualifiers
     source          1..6670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     primer_bind     717..736
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        717..735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      760..769
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2115..2134
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2797..3385
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2850..2870)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3268..3279)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3417..3893
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3616..3635)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3933..4073
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3933..4062
                     /label=AAV2 ITR
     rep_origin      4148..4603
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4235..4254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4445..4466
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4620..4639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4739..4761
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4799..4817)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4885..4989
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4990..5058,5059..5850)
                     /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(5208..5227)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6021..6609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6510..6529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ggacgaataa cgcgtggatc
      661 caccggtgag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      721 acgactcact atagggagac ccaagctggc tagcgtgccg ccaccatggc ctcgagaatg
      781 gagaccgaca ccctgctgct gtgggtgctg ctgctgtggg tgcccggctc caccggcgac
      841 agatctatgt tctctcccat ggcagtggcc caggctcgac gctataagtg gaggattcaa
      901 accgcgtggg atgctggaac agtaggctac agtctgttcc aaaagtttac ggagcgggta
      961 aaggaactga cggatggaca gcttgaggtc cagcctcttc cagcgggtgc ggtcgtaggc
     1021 acgtttgata tgttcgacgc ggtcaagact ggagttttgg acggaatgaa tcctttcacg
     1081 atctattggg cagagaggat gccggttaca gctttcctct ccagctacgc cctcgggttg
     1141 gaccgacctg accagtggga aacatggttt tactccctgg gtggcctcga caacgcccga
     1201 cgagcctttg ccgagcaagg cctcttctac gtgggaccag ttcagcacga cctcaataca
     1261 attcattcaa agaagccgat ccggagattt gaggacttca aaggtgtcaa gctccgagtg
     1321 ccaggcggca tgattgcgga agtctttgcg gccgcaggag cttccaccgt cctcttgcct
     1381 gggggggagg tctaccccgc tctggaaaga ggggcagtga ctgcggtttc cgagcggatg
     1441 taccccgagg acggcgccct gaagagcgag atcaagaagg ggctgaggct gaaggacggc
     1501 ggccactacg ccgccgtggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     1561 ggcgcctacg tcgtcgacat ccagttggac atcgtgtccc acaacgagga ctacactatc
     1621 gtggaacagt gcgaacgcgc cgagggccgc cactccaccg gcggcgtggt cgagctggac
     1681 aagggaggta caggcgggag tctggtgagc aagggcgagg aggatgacat ggccatcgtc
     1741 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     1801 atcgagggcg agggcgaggg ccgcccctac gaggcctttc agaccgctaa gctgaaggtg
     1861 accaagggtg gccccctgcc cttcgcctgg gacatcctat cccctcagtt catgtacggc
     1921 tccaaggcct acattaagca cccagccgac atccccgact acttcaagct gtccttcccc
     1981 gagggcttca ggtgggagcg cgtgatgtac ttcgaggacg gcggcattat tcacgttgac
     2041 caggactcct ccctgcagga cggcgtattc atctacaagg tgaagctgcg cggcaccaac
     2101 ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggagcg ggggcgtagt
     2161 gcggccgatt ttgttgggcc tgcagtaaac tataacttgg ggttccatca ggaggcaaag
     2221 tacatcataa tggggcctcc agagacgcct gccattcatc aacctgtaga ccttatggac
     2281 ttcaccataa accttaatcg gtggcgcagc cttccaaagc ctctgcaaga aagattcatc
     2341 gcggcggttc acgagtactc ttggattcac tacgcaggca ttcagaaggc taatttggag
     2401 gcgtggccga aatatcggca ggcaggtgtt gaggtcatcc ggctgggcaa cgaggatgtt
     2461 agaaaattca ggcgactcgc tatccccatt tggtttaagt gggctaagat ggataaatac
     2521 tcacgagagg ctttcgcgtc ccaactggag tatatgagag gtataggtca cgttacggac
     2581 gaggaattga agggcttgag cctgggctcc acctccggct ccggcaagcc cggctccggc
     2641 gagggctcca ccaagggcga attctcagat aacgactacg gtaaagatgg gttggctggg
     2701 gcttccggag gcagacgaaa tggcgctata actgtgctca gtttcataac gttctacgtg
     2761 gcagcattta tggtattgtt gtagaagctt atcgataatc aacctctgga ttacaaaatt
     2821 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     2881 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     2941 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3001 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3061 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3121 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3181 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg
     3241 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3301 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     3361 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt
     3421 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     3481 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     3541 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     3601 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     3661 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     3721 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     3781 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     3841 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     3901 tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     3961 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4021 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4081 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4141 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4201 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4261 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4321 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4381 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4441 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     4501 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     4561 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4621 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4681 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4741 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4801 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4861 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4921 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4981 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5041 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5101 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5161 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5221 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5281 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5341 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5401 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5461 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5521 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5581 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5641 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5701 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5761 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5821 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5881 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5941 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6001 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6061 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6121 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6181 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6241 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6301 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6361 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6421 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6481 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6541 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6601 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6661 gctcacatgt
//
