LOCUS       sequence-408947-        7360 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 7360)
  AUTHORS   Nasu Y, Aggarwal A, Le GNT, Vo CT, Kambe Y, Wang X, Beinlich FRM, 
            Lee AB, Ram TR, Wang F, Gorzo KA, Kamijo Y, Boisvert M, Nishinami S,
            Kawamura G, Ozawa T, Toda H, Gordon GR, Ge S, Hirase H, Nedergaard 
            M, Paquet ME, Drobizhev M, Podgorski K, Campbell RE
  TITLE     Lactate biosensors for spectrally and spatially multiplexed 
            fluorescence imaging.
  JOURNAL   Nat Commun. 2023 Oct 27;14(1):6598. doi: 10.1038/s41467-023-42230-5.
  PUBMED    37891202
REFERENCE   2  (bases 1 to 7360)
  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: "2023-10-27"; volume: "14(1):6598. doi"; pages: " 
            10.1038/s41467-023-42230-5"
COMMENT     Sequence Label: pcDNA-HA-eLACCO2.1-NGR
FEATURES             Location/Qualifiers
     source          1..7360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     644..664
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     738..757
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      781..790
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1771..1792)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2032..2051)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2826..2843)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2832..3056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3102..3530
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3189..3208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3399..3420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3539..3559)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3544..3873
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3724..3859
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3786..3805
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3940..4734
                     /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(3994..4013)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4604..4623
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4908..5029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4945..4964)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4999..5018
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5078..5094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5078..5094)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5091..5113)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5102..5118
                     /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(5126..5132,5133..5150,5151..5156))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5171..5192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5309..5326)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5480..6068)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5560..5579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6239..7030,7031..7099))
                     /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     6862..6881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7100..7204)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7279..7298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      721 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgtgcc
      781 gccaccatgg cctcgagaat gaagaccatc atcgccctgt cctacatctt ctgcctggtg
      841 ctgggcagat ctatgttctc tcccttggca gtggcccagg ctcgacgcta taggtggcgc
      901 attcaaaccg cgtgggatgc cggaacagta ggctacagtc tgttccaaaa gtttacggag
      961 cgggtaaagg aactgacgga tggacagctt gaggtccagc cttttccagc gagtgcagtc
     1021 gtaggcacgt ttgatatgtt cgacgcggtc aagactggag ttttggacgg aatgaatcct
     1081 ttcacgatct attgggcagg gaggatgccg gttacagctt tcctctccag ctacgccctc
     1141 gggttggacc gacctgacca gtgggaaaca tggttttact ccctgggtgg cctcgacaac
     1201 gcccgacgag cctttgccga gcaaggcctc ttctacgtgg gaccagttca gcacgacctc
     1261 aatacaattc attcaaagaa gccgatccgg agatttgagg acttcaaagg tgtcaagctc
     1321 cgagtgccgg gcggcatgat tgcggaagtt tttgcggccg caggagcttc caccgtcctc
     1381 ttgcctgggg gggaggtcaa ccccgctctg gaaagagggg taatagattg gagccacaac
     1441 gtccatatca tggccgacaa gcagaggaac ggcatcaagg cgaacttcga gatccgccac
     1501 agcaccgagg acggcggcgt gcagctcgcc taccactacc agcagaacac ccccatcggc
     1561 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagaccaa acttacgaaa
     1621 gaccccaacg agaagcgcga tcacatggtc ctgctggagt acgtgaccgc cgccgggatc
     1681 actcgcggca tggacgagct gtacaagggc ggtaccggag ggagaatggt gagcaagggc
     1741 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1801 cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca cctccggcaa gctgtccctg
     1861 aagttcatca gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1921 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1981 aagtcggcca tgcccgaagg ctacatccag gagcgcacca tcttcttcaa ggacgacggc
     2041 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2101 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcataagct ggagtacagc
     2161 tttaacgatg gtggtgcggc tgattttgtt gggcctgcag taaactataa cttggggttc
     2221 catcaggtgg caaagtacat cataatgggg cctccagaga cgcctgccat tcatcaacct
     2281 gtagacctca tggacttcac cataaacctt aatcggtggc gcagccttcc aaagcccctg
     2341 caagaaagat tcatcgcggc ggttcacgag tactcttgga ttcactacgc aggcattcag
     2401 aaggctaatt tggaggcgtg gccgaaatat cggcaggcag gtgttgaggt catccggctg
     2461 agcaacgagg atgttagaaa attcaggcga ctcgctattc ccatttggtt taagtgggct
     2521 aagatggata actactcacg agaggcattc gcgtctcaac tggagtatat gaaaggtata
     2581 ggttacgtta cggacgagga attgaagggc ttgggcctgg gctccacctc cggctccggc
     2641 aagcccggct ccggcgaggg ctccaccaag ggcgaattcg caggtagtgg tgcatctgga
     2701 acaggagatg cagaaggatc aggtgccctt cctgcccttg catgtagtct ggctccactg
     2761 ggactggcat tggttctgtg gacggtgttg ggcccctgtt agaagcttgg taaaccgctg
     2821 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2881 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2941 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3001 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3061 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3121 attaagcgcg gcgggtgtgg tggttacgcg cagtgtgacc gctacacttg ccagcgccct
     3181 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3241 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3301 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3361 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3421 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3481 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3541 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3601 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3661 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3721 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3781 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3841 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3901 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3961 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4021 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4081 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     4141 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4201 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4261 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4321 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4381 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4441 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4501 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4561 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4621 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4681 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4741 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4801 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4861 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4921 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4981 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     5041 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5101 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5161 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5221 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5281 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5341 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5401 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5461 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5521 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5581 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5641 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctatagg tatctcagtt
     5701 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5761 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5821 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5881 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5941 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6001 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6061 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6121 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6181 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6241 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6301 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6361 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6421 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6481 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6541 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6601 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6661 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6721 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6781 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6841 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6901 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6961 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7021 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7081 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7141 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7201 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cacccgatcc
     7261 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7321 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
//
