LOCUS       sequence-476991-        5442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of lowest affinity red fluorescent pyruvate 
            biosensor ApplePy1Lowest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5442)
  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 5442)
  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-ApplePy1Lowest
FEATURES             Location/Qualifiers
     source          1..5442
                     /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     complement(3110..3127)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3383..3469
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3561..3588
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3607..3698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3699..3767,3768..4559)
                     /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(3917..3936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4730..5318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5219..5238
                     /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 ctccgatccg ctggtagaac tgctttccga tcacccggaa tcccaatatg
     1861 acttgctcga aacgcgccat gcgctggaag ggatcgcagc atattacgct gccttgtgca
     1921 gctcagatga agagaaagag aggatcagag agttgcatca tgcgattgag cttgcccaac
     1981 aaagtggtga tctcgatgcc gaaagtaacg ctgtacttca gtatcaaatc gcggtgaccg
     2041 aggcagcgca caatgtggtc cttttgcact tgttgagata tatggagcct atgctcgcac
     2101 agaatgtccg acataacttt gagctgctgt actctggtac ggtggtttcc gagcggatgt
     2161 tccccgagga cggcgccctg aagagcgagg tcaagaaggg gctgaggctg aaggacggcg
     2221 gccactacgc cgtggaggtc aagaccacct acaaggccaa gaagcccgtg cccctgcccg
     2281 gcgcctacat cgtcgacatc atgttggaca tcgtgtccca caacgaggac tacactatcg
     2341 tggaacagtt cgaacgcgcc gagggccgcc accccaccgg cggcctggac gagctgtaca
     2401 agggaggtac aggcgggagt ctggtaagca agggcgagga ggatgacatg gccatcgtca
     2461 aggagttcat gcgcttcaag gtgcacatgg agggctccgt taacggccac gagttcgaga
     2521 tcgaaggcga gggcgagggc cgcccctacg aggcctttca gactgctaag ctgaaggtga
     2581 ccaagggtgg ccccctgccc ttcgcctggg acatcttatc ccctcagttc atgtacggct
     2641 ccaaggccta cattaagcac ccagccgata tccccgacta cttcaagctg tccttccccg
     2701 agggcttcag ctgggagcgc gtgatgtact tcgaggacgg cggcattatt cacgttaacc
     2761 aggactcctc cctgcaggac ggcgtattta tctacaaggt gaagctgcgc ggcaccaact
     2821 tcccccccga cggccccgta atgcagatga agaccatggg ctgggagtcg ggttggcgac
     2881 gagagatgct gccgctcgtg gcctcccatc gcactcgcat atttgaagct attatggcag
     2941 gaaaaccgga ggaagccagg gaagcgtcac atagacactt ggcctttatt aaggaaatcc
     3001 tgctggaccg gtcaaggggg gaatcacgcc gcgaactctc attgagaagg ctggagcaaa
     3061 gagagaattg aaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     3121 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     3181 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     3241 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     3301 tcgctgggaa cctgcggtta tctgctgttc ttctgggttt ccgtctggtc actgccggtg
     3361 gcgttgggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3421 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     3481 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     3541 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     3601 aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3661 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3721 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3781 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3841 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3901 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     3961 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4021 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4081 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4141 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4201 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4261 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4321 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4381 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4441 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4501 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4561 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4621 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4681 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4741 tttttctgcg cgtaatctgc tgcttgcaaa caaaacaacc accgctacca gcggtggttt
     4801 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4861 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4921 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4981 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5041 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5101 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5161 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5221 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5281 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5341 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5401 attctgtgga taaccgtatt accgcctttg agtgagctga ta
//
