LOCUS       sequence-251613-        4644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  R49A-K77A.
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: CIT0.5m
FEATURES             Location/Qualifiers
     source          1..4644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     99..116
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     201..220
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        201..219
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             251..273
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             293..310
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             314..346
                     /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(350..358,359..373)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(389..391,392..904)
                     /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             1916..2167
                     /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(2226..2244)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2240..2287
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2384..2839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2471..2490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2681..2702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2865..2969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2970..3038,3039..3830)
                     /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(3188..3207)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4001..4589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4490..4509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
       61 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      121 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      181 ggatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
      241 tagaaataat tttgtttaac tttaagaagg agatatacat atgcggggtt ctcatcatca
      301 tcatcatcat ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga
      361 cgatgacgat aaggatccgg gcctcgaggt gagcaagggc gaggagctgt tcaccggggt
      421 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      481 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      541 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt
      601 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      661 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      721 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      781 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacatcagcc acaacgtcta
      841 tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat
      901 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
      961 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1021 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggggtacccg
     1081 tctgcattat caggtcgggc agcgggcgct gattcaggca atgcagattt cggcgatgcc
     1141 ggagctggtc gaagcggttc agaaacgcga tctcgccaga atcaaagcgc ttatcgaccc
     1201 catggcttcg ttctccgacg ccacctatat caccgtcggt gatgccaacg gccagcgcct
     1261 ctatcatgtt aatcctgatg aaatcggcgc ctcgatggaa ggcggcgata gcgatgaagc
     1321 attgttaaac gcgaaaagtt acgtgtcagt gcgcaaaggc tcgctgggct cctcgctgcg
     1381 cggtaaatca ccgattcagg acgagaccgg caaggtgatt ggcattgtgt cggtaggcta
     1441 taccatcgag caaactagta agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1501 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1561 tgccacctac ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc
     1621 ctggcccacc ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga
     1681 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1741 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1801 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1861 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa
     1921 gcagaagaac ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt
     1981 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2041 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2101 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2161 gtacaagtaa gggcccgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     2221 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     2281 ttttttgctg aaaggaggaa ctatatccgg atctggcgta atagcgaaga ggcccgcacc
     2341 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     2401 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2461 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2521 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2581 gaccccaaaa aacttgatta gggtgttggt tcacgtagtg ggccatcgcc ctgatagacg
     2641 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2701 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2761 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2821 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2881 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2941 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3001 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3061 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3121 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3181 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3241 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3301 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3361 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3421 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3481 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3541 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3601 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3661 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3721 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3781 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3841 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3901 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3961 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4021 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4081 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4141 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4201 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4261 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4321 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4381 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4441 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4501 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4561 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4621 tggccttttg ctggcctttt gctc
//
