LOCUS       sequence-476574-        6634 bp    DNA     circular SYN 18-JUN-2025
DEFINITION  Mammalian expression of red fluorescent pyruvate control biosensor 
            dApplePy1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6634)
  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 6634)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 18, 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-dApplePy1
FEATURES             Location/Qualifiers
     source          1..6634
                     /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(2100..2117)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2106..2330
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2376..2804
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2463..2482)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2673..2694
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2813..2833)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2818..3147
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2998..3133
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3060..3079
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3214..4008
                     /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(3268..3287)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3878..3897
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4182..4303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4219..4238)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4273..4292
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4352..4368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4352..4368)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4365..4387)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4376..4392
                     /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(4400..4406,4407..4424,4425..4430))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4445..4466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4583..4600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4754..5342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4834..4853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5513..6304,6305..6373))
                     /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     6136..6155
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6374..6478)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6553..6572)
                     /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 ttctccgatc cgctgctaga actgctttcc
      841 gatcacccgg aatcccaata tgacttgctc gaaacgcgcc atgcgctgga agggatcgca
      901 gcatattacg ctgccttgtg cagctcagat gaagagaaag agaggatcag agagttgcat
      961 catgcgattg agcttgccca acaaagtggt gatctcgatg ccgaaagtaa cgctgtactt
     1021 cagtatcaaa tcgcggtgac cgaggcagcg cacaatgtgg tccttttgca cttgctgaga
     1081 tatatggagc ctatgctcgc acagaatatc cgacataact ttgagctgct gtactctggt
     1141 acggtggttt ccgagcggat gttccccgag gacggcgccc tgaagagcga ggtcaagaag
     1201 gggctgaggc tgaaggacgg cggccactac gccgtggagg tcaagaccac ctacaaggcc
     1261 aagaagcccg tgcccctgcc cggcgcctac atcgtcgaca tcatgttgga catcgtgtcc
     1321 cacaacgagg actacactat cgtggaacag ttcgaacgcg ccgagggccg ccaccccacc
     1381 ggcggcctgg acgagctgta caagggaggt acaggcggga gtctggtaag caagggcgag
     1441 gaggatgaca tggccatcgt caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1501 gttaacggcc acgagttcga gatcgaaggc gagggcgagg gccgccccta cgaggccttt
     1561 cagactgcta agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatctta
     1621 tcccctcagt tcatgtacgg ctccaaggcc tacattaagc acccagccga tatccccgac
     1681 tacttcaagc tgtccttccc cgagggcttc agctgggagc gcgtgatgta cttcgaggac
     1741 ggcggcatta ttcacgttaa ccaggactcc tccctgcagg acggcgtatt tatctacaag
     1801 gtgaagctgc gcggcaccaa cttccccccc gacggccccg taatgcagat gaagaccatg
     1861 ggctgggagt cgggttgggg acgacgagag atgctgccgc tcgtggcctc ccatcgcact
     1921 cgcatatttg aagctattat ggcaggaaaa ccggaggaag ccagggaagc gtcacataga
     1981 cacttggcct ttattaagga aatcctgctg gaccggtcaa ggggggaatc acgccgcgaa
     2041 ctctcattga gaaggctgga gcaaagagag aattgaaagc ttggtaaacc gctgatcagc
     2101 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2161 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2221 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2281 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2341 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2401 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2461 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2521 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2581 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2641 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2701 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2761 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     2821 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2881 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2941 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3001 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3061 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3121 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3181 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3241 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3301 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3361 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3421 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3481 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3541 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3601 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3661 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3721 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3781 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3841 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3901 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3961 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4021 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4081 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4141 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4201 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4261 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4321 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4381 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4441 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4501 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4561 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4621 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4681 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4741 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4801 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4861 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4921 tcccttcggg aagcgtggcg ctttctcata gctcacgcta taggtatctc agttcggtgt
     4981 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5041 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5101 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5161 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5221 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5281 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5341 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5401 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5461 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5521 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5581 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5641 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5701 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5761 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5821 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5881 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5941 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6001 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6061 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6121 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6181 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6241 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6301 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6361 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6421 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6481 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6541 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6601 ttgtgtgttg gaggtcgctg agtagtgcgc gagc
//
