LOCUS       sequence-251592-        4644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  R66A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4644)
  AUTHORS   Ewald JC, Reich S, Baumann S, Frommer WB, Zamboni N
  TITLE     Engineering genetically encoded nanosensors for real-time in vivo 
            measurements of citrate concentrations.
  JOURNAL   PLoS One. 2011;6(12):e28245. doi: 10.1371/journal.pone.0028245. Epub
            2011 Dec 2.
  PUBMED    22164251
REFERENCE   2  (bases 1 to 4644)
  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: "PLoS One.";
            date: "2011"; volume: "6(12):e28245. doi"; pages: " 
            10.1371/journal.pone.0028245. Epub 2011 Dec 2"
COMMENT     Sequence Label: CIT0
FEATURES             Location/Qualifiers
     source          1..4644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..114
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     199..218
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        199..217
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             249..271
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             291..308
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             312..344
                     /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(348..356,357..371)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(387..389,390..902)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKIRHNIE"
     CDS             1914..2165
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     complement(2224..2242)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2238..2285
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2382..2837
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2469..2488)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2679..2700
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2863..2967
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2968..3036,3037..3828)
                     /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(3186..3205)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3999..4587
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4488..4507
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
       61 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      121 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagg
      181 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
      241 gaaataattt tgtttaactt taagaaggag atatacatat gcggggttct catcatcatc
      301 atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg
      361 atgacgataa ggatccgggc ctcgaggtga gcaagggcga ggagctgttc accggggtgg
      421 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      481 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      541 agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca
      601 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
      661 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
      721 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
      781 aggacggcaa catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata
      841 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
      901 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
      961 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1021 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg ggtacccgtc
     1081 tgcattatca ggtcgggcag cgggcgctga ttcaggcaat gcagatttcg gcgatgccgg
     1141 agctggtcga agcggttcag aaacgcgatc tcgccagaat caaagcgctt atcgatccca
     1201 tgcgttcgtt ctccgacgcc acctatatca ccgtcggtga tgccagcggc caggccctgt
     1261 atcacgtcaa tcctgatgaa atcggcaaat cgatggaggg cggcgatagc gatgaagcat
     1321 tgttaaacgc gaaaagttac gtgtcagtgc gcaaaggctc gctgggctcc tcgctgcgcg
     1381 gtaaatcacc gattcaggac gagaccggca aggtgattgg cattgtgtcg gtaggctata
     1441 ccatcgagca aactagtaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1501 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1561 ccacctacgg caagctgacc ctgaagctga tctgcaccac cggcaagctg cccgtgccct
     1621 ggcccaccct cgtgaccacc ctgggctacg gcctgcagtg cttcgcccgc taccccgacc
     1681 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1741 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1801 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1861 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcacc gccgacaagc
     1921 agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc
     1981 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2041 acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2101 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2161 acaagtaagg gcccgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     2221 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2281 ttttgctgaa aggaggaact atatccggat ctggcgtaat agcgaagagg cccgcaccga
     2341 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     2401 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2461 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2521 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2581 ccccaaaaaa cttgattagg gtgttggttc acgtagtggg ccatcgccct gatagacggt
     2641 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2701 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2761 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2821 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2881 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2941 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3001 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3061 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3121 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3181 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3241 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3301 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3361 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3421 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3481 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3541 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3601 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3661 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3721 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3781 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3841 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3901 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3961 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4021 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4081 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4141 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4201 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4261 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4321 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4381 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4441 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4501 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4561 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4621 gccttttgct ggccttttgc tcac
//
