LOCUS       sequence-316042-        6871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses biosensors for pyruvate in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6871)
  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 6871)
  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 Pegassos
FEATURES             Location/Qualifiers
     source          1..6871
                     /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"
     primer_bind     complement(1233..1252)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2372..2393
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2465..2482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2471..2695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2741..3169
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2828..2847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3038..3059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3178..3198)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3183..3512
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3363..3498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3425..3444
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3579..4373
                     /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(3633..3652)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4243..4262
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4547..4668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4584..4603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4638..4657
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4717..4733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4717..4733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4730..4752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4741..4757
                     /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(4765..4771,4772..4789,4790..4795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4810..4831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4948..4965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5119..5704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5199..5218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5875..6666,6667..6735))
                     /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     6498..6517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6736..6840)
                     /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 gtttaaacgg gccctctaga ctcgagatgg tgagcaaggg cgaggagctg ttcaccgggg
      961 tggtgcccat ccaggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1021 gcgagggtga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1081 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1141 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1201 gctacatcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1261 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1321 aggaggacgg caacatcctg gggcacaagc tggagtacaa cgcgggtacg actctttaca
     1381 gcaaaatccg ccaaccaaaa ctctccgatg tgattgagca gcaactggag tttttgatcc
     1441 tcgaaggcac tctccgcccg ggcgaaaaac tcccaccgga acgcgaactg gcaaaacagt
     1501 ttgacgtctc ccgtccctcc ttgcgtgagg cgattcaacg tctcgaagcg aagggcttgt
     1561 tgcttcgtcg ccagggtggc ggcacttttg tccagagcag cctatggcaa agtttcagcg
     1621 atccgctggt ggagctgctc tccgaccatc ctgagtcaca gtatgacttg ctcgaaacac
     1681 gacacgccct ggaaggtatc gccgcttatt acgccgcgct gcgtagtacc gatgaagaca
     1741 aggaacgcat ccgtgaactc caccacgcca tagagctggc gcagcagtct ggcgatctgg
     1801 acgcggaatc aaacgccgta ctccagtatc agattgccgt caccgaagcg gcccacaatg
     1861 tggttctgct tcatctgcta aggtgtatgg agccgatgtt ggcccagaat gtccgccaga
     1921 acttcgaatt gctctattcg cgtcgcgaga tgctgccgct ggtgagtagt caccgcaccc
     1981 gcatatttga agcgattatg gccggtaagc cggaagaagc gcgcgaagca tcgcatcgcc
     2041 atctggcctt tatcgaagaa attttgctcg acagaagtcg tgaagagagc cgccgtgagc
     2101 gttctctgcg tcgtctggag caacgaaagc ctcttaagcg gtcgggttta acgtcgtcac
     2161 tggagaacgt ctatatcaag gccgacaagc agaagaacgg catcaaggcg aacttcaaga
     2221 tccgccacaa catcgaggac ggcggcgtgc agctcgccta ccactaccag cagaacaccc
     2281 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcgtg cagtccatac
     2341 tttcgaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2401 ccgggatcac tctcggcatg gacgagctgt acaagtaaaa gcttaagttt aaaccgctga
     2461 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2521 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2581 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2641 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2701 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2881 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2941 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3001 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3061 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3121 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     3181 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3241 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3301 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3361 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3421 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3481 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3541 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3601 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3661 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3721 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3781 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3841 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3901 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3961 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4021 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4081 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     4141 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4201 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4261 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4321 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4381 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4681 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4741 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4801 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4861 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4921 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5101 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5161 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5221 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5281 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5341 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5401 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5461 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5521 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5581 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5641 caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5701 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5761 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5821 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5881 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5941 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6001 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6061 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6121 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6181 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6241 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6301 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6361 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6421 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6481 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6541 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6601 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6661 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6721 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6781 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6841 cacatttccc cgaaaagtgc cacctgacgt c
//
