LOCUS       sequence-431764-        5345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of genetically encoded green fluorescent 
            L-lactate biosensor with high affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5345)
  AUTHORS   Hario S, Le GNT, Sugimoto H, Takahashi-Yamashiro K, Nishinami S, 
            Toda H, Li S, Marvin JS, Kuroda S, Drobizhev M, Terai T, Nasu Y, 
            Campbell RE
  TITLE     High-Performance Genetically Encoded Green Fluorescent Biosensors 
            for Intracellular l-Lactate.
  JOURNAL   ACS Cent Sci. 2024 Jan 31;10(2):402-416. doi: 
            10.1021/acscentsci.3c01250. eCollection 2024 Feb 28.
  PUBMED    38435524
REFERENCE   2  (bases 1 to 5345)
  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"; doi: 
            "10.1021/acscentsci.3c01250"; journalName: "ACS Cent Sci"; date: 
            "2024-01-31- 31"; volume: "10"; issue: "2"; pages: "402-416"
COMMENT     Sequence Label: pBAD-iLACCO1.2
FEATURES             Location/Qualifiers
     source          1..5345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /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     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             331..348
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             352..384
                     /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(388..396,397..411)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     963..984
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1090..1111)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1351..1370)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(1746..1763)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1916..2002
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2094..2121
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2140..2231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2232..2300,2301..3092)
                     /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(2450..2469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3263..3851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3752..3771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4005..4022
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4037..4177
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4103..4122)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4263..4285
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4441..5319)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat ggggggttct catcatcatc atcatcatgg tatggctagc
      361 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgagc
      421 tcgagaatgg aacaaaacat agtccagccg ttgaagaccc acatggcaga cgaccctgat
      481 tatacatttg atattctgga ggcgcggtac gcgatagaag cgtcaactgc ctggcacgct
      541 gcaatgcgag ctacacccgg ggacaaagag aaaattcaac tgtgcttcga ggctacactg
      601 tccgaagatc ccgatatagc ttcccaagca gacgtaaggt tccacctggc gattgctgaa
      661 gcaagccata acatagttct gcttcagaca atgcgaggct attttgacgt tttgcagtct
      721 tcaattaggc acagtcgaca gcggatgtac aggaagaacg tctatatcat ggccgacaag
      781 cagaggaacg gcatcaaggc gaacttcatg atccgccaca acatcgaggg cggcggcgtg
      841 cagctcgcct accactacca gcagaacgcc cccatcggcg acggccccgt gctgctgccc
      901 gacagccact acctgagcac ccagtccaaa ctttcgaaag accccaacga gaagcgcgat
      961 cacatggtcc tgctggagtt cgtgaccgcc gccgggacca ctcccggcat ggacgagctg
     1021 tacaagggcg gtaccagagg gagcatggtg agcaagggcg aggagctgtt caccggggtg
     1081 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1141 gagggtgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1201 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1261 ggccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1321 tacatccagg agcgctccat cttcttcgag gacgacggca actacaagac ccgcgccgag
     1381 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1441 gaggacggca acatcctggg gcataagctg gagtacaacc ttgaggaggg tgtgccgccg
     1501 gtcttcagtc aactcacaga acagcatcag gcagttatag acgccatctt tgccggcgat
     1561 gcggacggtg caagaaaagc tatgttggct catctgagct tcgtccatac gactatgaaa
     1621 cgcttcgacg atgatcatgc ccgacacgca cgcattacca gactgcctgg agagcataat
     1681 gagcactctc gggaaaagaa tgcatgaaag cttggctgtt ttggcggatg agagaagatt
     1741 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     1801 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     1861 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     1921 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     1981 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     2041 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     2101 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     2161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2221 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2281 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2341 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2401 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2461 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2521 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2581 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2641 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2701 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2761 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2821 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2881 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2941 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3001 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3061 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3301 aaacaaaaca accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3901 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3961 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4021 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4081 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4141 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4201 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4261 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagca
     4321 gatcaattcg cgcgcgaagg cgaagcggca tgcataatgt gcctgtcaaa tggacgaagc
     4381 agggattctg caaaccctat gctactccgt caagccgtca attgtctgat tcgttaccaa
     4441 ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct
     4501 cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc
     4561 aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg
     4621 gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag
     4681 atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt
     4741 gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg
     4801 tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt
     4861 tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa
     4921 caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa
     4981 tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg
     5041 gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa
     5101 cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg
     5161 accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa
     5221 atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga
     5281 gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt
     5341 cagag
//
