LOCUS       sequence-476342-        6622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of lowest affinity green fluorescent pyruvate 
            biosensor GreenPy1Lowest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6622)
  AUTHORS   Imai S, Hario S, Suerte C, Yamaguchi I, Sakoi K, Terai T, 
            Takahashi-Yamashiro K, Campbell RE
  TITLE     High-performance genetically-encoded green and red fluorescent 
            biosensors for pyruvate
  JOURNAL   bioRxiv 2025.04.17.649293
REFERENCE   2  (bases 1 to 6622)
  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: "bioRxiv 
            2025.04.17.649293"
COMMENT     Sequence Label: pcDNA-GreenPy1Lowest
FEATURES             Location/Qualifiers
     source          1..6622
                     /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     1347..1368
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1474..1495)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1735..1754)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2088..2105)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2094..2318
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2364..2792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2451..2470)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2661..2682
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2801..2821)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2806..3135
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2986..3121
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3048..3067
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3202..3996
                     /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(3256..3275)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3866..3885
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4170..4291
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4207..4226)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4261..4280
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4340..4356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4340..4356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4353..4375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4364..4380
                     /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(4388..4394,4395..4412,4413..4418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4433..4454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4571..4588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4742..5330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4822..4841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5501..6292,6293..6361))
                     /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     6124..6143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6362..6466)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6541..6560)
                     /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 ggtgcaatct ttttccgatc cgctggtaga actgcttcca
      841 gatcacccgg aatcccaata tgacttgctc gaagcgcgcc atgcgctgga agggatcgca
      901 gcatattacg ctgccttgcg cagcacagat gaagataaag agaggatcag agagttgcat
      961 catgcgattg ggcttgccca acaaagtggt gatctcgatg ccgaaagtaa cgctgtactt
     1021 cagtatcaaa ccgcggtgac cgaggcagcg cacaatgtgg tccttttgca cttgttgaga
     1081 tgtatggagc ctatgctcgc acagaatgtc cgacaaaact ttgagctgct gtaccgtcat
     1141 aacgtctatg tcatggccga caagcagagg aacggcatca aggcgaactt caagatccgc
     1201 cacaacatcg agggcggcgg cgtgcagctc gcctaccacc accagcagaa cacccccatc
     1261 ggcgacggcc ccgtgctgct gcccgacaac cactacctgt gcacccagtc caaactttcg
     1321 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1381 atcactctcg gcatggacga gctgtacatg ggcggtaccg gagggagcat ggtgagcaag
     1441 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1501 ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc
     1561 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1621 ctgacctacg gcgtgcagtg cttcggccgc taccccgacc acatgaagca gcacgacttc
     1681 ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt cgaggacgac
     1741 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gagccgcatc
     1801 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcataa gctggagtac
     1861 aactcgtggc gacgagagat gctgccgctc gtgtcttccc atcgcactcg catatttgaa
     1921 gctattatgg caggaaaacc ggaggaagcc agggaagcgt cccataaaca cttggccttt
     1981 attgaggaaa tcctgctgga ccggtcaagg gaggaatcac gccgcgaacg ctcattgaga
     2041 cggctggagc aaagaaagaa ttgaaagctt ggtaaaccgc tgatcagcct cgactgtgcc
     2101 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2161 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2221 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2281 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2341 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2401 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2461 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2521 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2581 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2641 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2701 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2761 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2821 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2881 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2941 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3001 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3061 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3121 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3181 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3241 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3301 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3361 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3421 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3481 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3541 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3601 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3661 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3721 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3781 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3841 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3901 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3961 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4021 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4081 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4141 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4201 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4261 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4321 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4381 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4441 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4501 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4561 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4621 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4681 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4741 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4801 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4861 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4921 gcgtggcgct ttctcatagc tcacgctata ggtatctcag ttcggtgtag gtcgttcgct
     4981 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5041 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5101 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5161 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5221 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5281 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5341 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5401 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5461 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6361 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6481 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     6541 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     6601 ggtcgctgag tagtgcgcga gc
//
