LOCUS       sequence-476976-        5333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of green fluorescent pyruvate control biosensor
            dGreenPy1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5333)
  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 5333)
  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: pBAD-dGreenPy1
FEATURES             Location/Qualifiers
     source          1..5333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..47
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    62..202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(128..147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     288..310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(466..1344)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1371..1655
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1578..1597
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1701..1718
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1722..1754
                     /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="MASMTGGQQMG"
     CDS             join(1758..1766,1767..1781)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     2345..2366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2472..2493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2733..2752)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3104..3121)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3274..3360
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3452..3479
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3498..3589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3590..3658,3659..4450)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3808..3827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4621..5209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5110..5129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
       61 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
      121 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
      181 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
      241 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
      301 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagcagatca attcgcgcgc
      361 gaaggcgaag cggcatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac
      421 cctatgctac tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac
      481 ggctacatca ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt
      541 gcatttttta aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac
      601 ggtggcgata ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc
      661 ctcgcgccag cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga
      721 cggcgacaag caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg
      781 atcgctgatg tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc
      841 gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc
      901 cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg
      961 cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt
     1021 aagccattca tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg
     1081 agcctccgga tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc
     1141 cggtcggcaa acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag
     1201 attgagaata taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt
     1261 ggcctcaatc ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag
     1321 gggatcattt tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa
     1381 ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac
     1441 caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac
     1501 aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca
     1561 cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg
     1621 acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc taacaggagg
     1681 aattaaccat ggggggttct catcatcatc atcatcatgg tatggctagc atgactggtg
     1741 gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgagc tcgagaatgg
     1801 tgcaatcttt ttccgatccg ctggtagaac tgcttccaga tcacccggaa tcccaatatg
     1861 acttgctcga agcgcgccat gcgctggaag ggatcgcagc atattacgct gccttgcgca
     1921 gcacagatga agataaagag aggatcagag agttgcatca tgcgattggg cttgcccaac
     1981 aaagtggtga tctcgatgcc gaaagtaacg ctgtacttca gtatcaaacc gcggtgaccg
     2041 aggcagcgca caatgtggtc cttttgcact tgttgagatg tatggagcct atgctcgcac
     2101 agaatatccg acaaaacttt gagctgctgt accgtcataa cgtctatgtc atggccgaca
     2161 agcagaggaa cggcatcaag gcgaacttca agatccgcca caacatcgag ggcggcggcg
     2221 tgcagctcgc ctaccaccac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2281 ccgacaacca ctacctgtgc acccagtcca aactttcgaa agaccccaac gagaagcgcg
     2341 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2401 tgtacatggg cggtaccgga gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg
     2461 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2521 gcgagggtga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2581 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2641 tcggccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2701 gctacatcca ggagcgcacc atcttcttcg aggacgacgg caactacaag acccgcgccg
     2761 aggtgaagtt cgagggcgac accctggtga gccgcatcga gctgaagggc atcgacttca
     2821 aggaggacgg caacatcctg gggcataagc tggagtacaa ctcgtggggt cgacgagaga
     2881 tgctgccgct cgtgtcttcc catcgcactc gcatatttga agctattatg gcaggaaaac
     2941 cggaggaagc cagggaagcg tcccataaac acttggcctt tattgaggaa atcctgctgg
     3001 accggtcaag ggaggaatca cgccgcgaac gctcattgag acggctggag caaagaaaga
     3061 attgaaagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     3121 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     3181 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     3241 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     3301 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3361 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     3421 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     3481 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3541 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3601 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3661 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3721 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3781 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     3841 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3901 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3961 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4021 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4081 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4141 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4201 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4261 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4321 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4381 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4441 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4501 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4561 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4621 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaacaac caccgctacc
     4681 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4741 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     4801 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4861 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4921 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4981 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5041 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5101 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5161 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5221 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5281 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ata
//
