LOCUS       sequence-446185-        4025 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of N-terminal His-tagged qmTQ2-citrate, a cyan 
            fluorescent FLIM biosensor for detecting citrate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4025)
  AUTHORS   Zhong C, Arai S, Okada Y
  TITLE     Development of fluorescence lifetime biosensors for ATP, cAMP, 
            citrate, and glucose using the mTurquoise2-based platform.
  JOURNAL   Cell Rep Methods. 2024 Nov 18;4(11):100902. doi: 
            10.1016/j.crmeth.2024.100902.
  PUBMED    39561716
REFERENCE   2  (bases 1 to 4025)
  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: "Cell Rep 
            Methods."; date: "2024-11-18"; volume: "4(11):100902. doi"; pages: "
            10.1016/j.crmeth.2024.100902"
COMMENT     Sequence Label: pRSETA-qmTQ2-citrate
FEATURES             Location/Qualifiers
     source          1..4025
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             133..165
                     /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(169..177,178..192)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(262..283)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(523..542)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1114..1365
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     1302..1323
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1426..1444)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1440..1487
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1584..2039
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1671..1690)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1881..1902
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2065..2169
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2170..2238,2239..3030)
                     /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     complement(2388..2407)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3201..3789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3690..3709
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3943..3960
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcggggttc tcatcatcat
      121 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
      181 gatgacgata aggatcgatg gggatccgag ctcgagatgg tgagcaaggg cgaggagctg
      241 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      301 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
      361 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gtcctggggc
      421 gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
      481 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
      541 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
      601 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacggtacc
      661 gcactgaaga aggaaaccga ggagcgtctg cattatcagg tcgggcaacg ggcgctgatt
      721 caggcgatgc agatttcggc gatgccggag ctggtggagg cagtgcagaa acgcgatctc
      781 gccagaatca aagcccttat cggccccatg cgttcgttct ccgacgccac ctacatcacc
      841 gtcggtgatg ccagcggcca gcgcctctat cacgtcaatc ctgatgaaat cggcaaatcg
      901 atggaaggcg gcgatagcga tgaggcgttg ataaacgcta aaagctacgt gtcagtgcgc
      961 aaaggctcgc tgggatcgtc gctgcgcggt aaatcgccga ttcaggacgc aaccggcaag
     1021 gtgatcggca ttgtgtcggt aggctatacc attgagcaac tggaagcact gaagaaagaa
     1081 gaattcttca gcgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1141 aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag
     1201 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1261 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1321 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaacg aagcttgatc
     1381 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1441 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1501 ctatatccgg atctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1561 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1621 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1681 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1741 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1801 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1861 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1921 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1981 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2041 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2101 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2161 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2221 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2281 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2341 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2401 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2461 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2521 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2581 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2641 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2701 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2761 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2821 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2881 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2941 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3001 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3061 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3121 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3181 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3241 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3301 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3361 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3421 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3481 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3541 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3601 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3661 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3721 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3781 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3841 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3901 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3961 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4021 tgcag
//
