LOCUS       sequence-321693-        6874 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 6874)
  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 6874)
  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..6874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(999..1018)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2138..2159
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2231..2248)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2237..2461
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2507..2935
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2594..2613)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2804..2825
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2944..2964)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2949..3278
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3129..3264
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3191..3210
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3345..4139
                     /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(3399..3418)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4009..4028
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4313..4434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4350..4369)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4404..4423
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4483..4499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4483..4499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4496..4518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4507..4523
                     /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(4531..4537,4538..4555,4556..4561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4576..4597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4714..4731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4885..5473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4965..4984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5644..6435,6436..6504))
                     /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     6267..6286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6505..6609)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6684..6703)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacgggccc tctagactcg agatggtgag caagggcgag gagctgttca
      721 ccggggtggt gcccatccag gtcgagctgg acggcgacgt aaacggccac aagttcagcg
      781 tgtccggcga gggtgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
      841 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
      901 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
      961 ccgaaggcta catccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1021 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1081 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacgcg ggtacgactc
     1141 tttacagcaa aatccgccaa ccaaaactct ccgatgtgat tgagcagcaa ctggagtttt
     1201 tgatcctcga aggcactctc cgcccgggcg aaaaactccc accggaacgc gaactggcaa
     1261 aacagtttga cgtctcccgt ccctccttgc gtgaggcgat tcaacgtctc gaagcgaagg
     1321 gcttgttgct tcgtcgccag ggtggcggca cttttgtcca gagcagccta tggcaaagtt
     1381 tcagcgatcc gctggtggag ctgctctccg accatcctga gtcacagtat gacttgctcg
     1441 aaacacgaca cgccctggaa ggtatcgccg cttattacgc cgcgctgcgt agtaccgatg
     1501 aagacaagga acgcatccgt gaactccacc acgccataga gctggcgcag cagtctggcg
     1561 atctggacgc ggaatcaaac gccgtactcc agtatcagat tgccgtcacc gaagcggccc
     1621 acaatgtggt tctgcttcat ctgctaaggt gtatggagcc gatgttggcc cagaatgtcc
     1681 gccagaactt cgaattgctc tattcgcgtc gcgagatgct gccgctggtg agtagtcacc
     1741 gcacccgcat atttgaagcg attatggccg gtaagccgga agaagcgcgc gaagcatcgc
     1801 atcgccatct ggcctttatc gaagaaattt tgctcgacag aagtcgtgaa gagagccgcc
     1861 gtgagcgttc tctgcgtcgt ctggagcaac gaaagcctct taagcggtcg ggtttaacgt
     1921 cgtcactgga gaacgtctat atcaaggccg acaagcagaa gaacggcatc aaggcgaact
     1981 tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgcctaccac taccagcaga
     2041 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcgtgcagt
     2101 ccatactttc gaaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2161 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaaagctt aagtttaaac
     2221 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2281 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2341 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2401 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2461 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     2521 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2581 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2641 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2701 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2761 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2821 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2881 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     2941 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3001 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3061 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3121 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3181 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3241 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3301 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3361 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3421 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3481 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3541 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3601 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3661 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3721 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3781 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3841 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3901 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3961 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4021 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4081 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4141 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4201 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4261 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4321 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4381 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4441 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4501 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4561 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4621 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4681 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4741 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4801 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4861 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4921 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4981 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5041 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5101 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5161 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5221 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5281 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5341 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5401 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5461 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5521 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5581 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5641 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5701 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5761 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5821 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5881 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5941 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6001 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6061 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6121 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6181 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6241 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6301 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6361 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6421 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6481 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6541 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6601 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     6661 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     6721 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     6781 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     6841 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
