LOCUS       sequence-316038-        6986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses biosensor for lactate in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  AUTHORS   Harada K, Chihara T, Hayasaka Y, Mita M, Takizawa M, Ishida K, Arai 
            M, Tsuno S, Matsumoto M, Ishihara T, Ueda H, Kitaguchi T, Tsuboi T
  TITLE     Green fluorescent protein-based lactate and pyruvate indicators 
            suitable for biochemical assays and live cell imaging.
  JOURNAL   Sci Rep. 2020 Nov 11;10(1):19562. doi: 10.1038/s41598-020-76440-4.
  PUBMED    33177605
REFERENCE   2  (bases 1 to 6986)
  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: "Sci Rep."; 
            date: "2020-11-11"; volume: "10(1):19562. doi"; pages: " 
            10.1038/s41598-020-76440-4"
COMMENT     Sequence Label: Green Lindoblum
FEATURES             Location/Qualifiers
     source          1..6986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             895..924
                     /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="ASMTGGQQMG"
     CDS             join(928..936,937..951)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     2487..2508
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2580..2597)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2586..2810
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2856..3284
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2943..2962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3153..3174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3293..3313)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3298..3627
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3478..3613
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3694..4488
                     /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(3748..3767)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4358..4377
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4662..4783
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4699..4718)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4753..4772
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4832..4848)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4832..4848)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4845..4867)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4856..4872
                     /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(4880..4886,4887..4904,4905..4910))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4925..4946
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5063..5080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5234..5819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5314..5333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5990..6781,6782..6850))
                     /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     6613..6632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6851..6955)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatcgatgg
      961 ggatctgccg aggaaagcga cagcgtggat tctgcggatg cagaggagga tgactctgat
     1021 gtctggtggg gtggagcgga cacagactac gctgatggcg gtgaagacaa agtagtagaa
     1081 gtcgccgaag aggaggaagt ggctgatgtt gaggaagagg aagctgatga tgatgaggat
     1141 gtggaggatg gggacgaggt ggaggaggag gccgaggagc cctacgaaga ggccaccgag
     1201 agaacaacca gcactgccac caccaccaca accaccactg agtccgtgga ggaggtggga
     1261 tccatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcca ggtcgagctg
     1321 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggtgaggg cgatgccacc
     1381 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1441 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1501 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acatccagga gcgcaccatc
     1561 ttcttcaagg acgacggaaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1621 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1681 cacaagctgg agtatactgg taagtttgtg cagcgtctga tggccgatga tccggattac
     1741 agtttcgata ttctggaagc ccgctacgcc attgaagcca gcaccgcatg gcatgcggca
     1801 atgcgcgcca cacctggcga caaagaaaag attcagcttt gctttgaagc aacgctaagt
     1861 gaagacccgg atatcgcctc acaagcggac gttcgttttc atctggcgat tgccgaagcc
     1921 tcacataaca tcgtgctgct gcaaaccatg cgcggtttct tcgatgtcct gcaatcctca
     1981 gtgaagcata gccgtcagcg gatgtatctg gtgccaccgg ttttttcaca actgaccgaa
     2041 caacatcagg ctgtcattga cgccattttt gccggtgatg ctgacggggc gcgtaaagca
     2101 atgatggcgc accttagttt tgttcacacc accatgaaac gattcgatga agatcaggct
     2161 cgccacgcac ggattacccg cctgcccgct gagcataatg agcattcgag ggagaaaaac
     2221 gcacttaagg ctagggagtg gttattggca ttaaagttgg aattagccag gtcactggag
     2281 aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc
     2341 cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc
     2401 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc catactttcg
     2461 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2521 atcactctcg gcatggacga gctgtacaag taaaagctta agtttaaacc gctgatcagc
     2581 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2641 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2701 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2761 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2821 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2881 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2941 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3001 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3061 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3121 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3181 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3241 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3301 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3361 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3421 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3481 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3541 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3601 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3661 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3721 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3781 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3841 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3901 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3961 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4021 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4081 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4141 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4201 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4261 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4321 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4381 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4441 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4501 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4561 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4621 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4681 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4741 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4801 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4861 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4921 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4981 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5041 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5101 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5161 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5221 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5281 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5341 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5401 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5461 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5521 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5581 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5641 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5701 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5761 ctggtagcgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5821 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5881 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5941 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6001 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6061 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6121 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6181 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6241 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6301 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6361 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6421 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6481 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6541 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6601 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6661 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6721 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6781 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6841 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6901 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6961 ttccccgaaa agtgccacct gacgtc
//
