LOCUS       sequence-476335-        5339 bp    DNA     circular SYN 18-JUN-2025
DEFINITION  Bacterial expression of high affinity red fluorescent pyruvate 
            biosensor ApplePy1High.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5339)
  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 5339)
  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: pBAD-ApplePy1High
FEATURES             Location/Qualifiers
     source          1..5339
                     /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      3280..3366
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3458..3485
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3504..3595
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3596..3664,3665..4456)
                     /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(3814..3833)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4627..5215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5116..5135
                     /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 ctgctagaac 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 tgctgagata tatggagcct atgctcgcac
     2101 agaatatccg 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 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     3361 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     3421 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     3481 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     3541 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3601 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3661 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3721 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3781 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     3841 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3901 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3961 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4021 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4081 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4141 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4201 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4261 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4321 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4381 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4441 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4501 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4561 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4621 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aacaaccacc
     4681 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4741 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4801 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4861 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4921 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4981 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5041 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5101 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5161 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5221 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5281 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgata
//
