LOCUS       sequence-7677-ex        5121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5121)
  AUTHORS   Deuschle K, Okumoto S, Fehr M, Looger LL, Kozhukh L, Frommer WB
  TITLE     Construction and optimization of a family of genetically encoded 
            metabolite sensors by semirational protein engineering.
  JOURNAL   Protein Sci. 2005 Sep . 14(9):2304-14.
  PUBMED    16131659
REFERENCE   2  (bases 1 to 5121)
  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: "Protein 
            Sci. 2005 Sep . 14(9):2304-14."
COMMENT     Sequence Label: pRSET FLIPE intramol-1m
FEATURES             Location/Qualifiers
     source          1..5121
                     /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"
     CDS             join(301..303,304..306,307..819)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIE"
     CDS             join(1747..1749,1750..1752,1753..2466)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2522..2540)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2536..2583
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2680..3135
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2767..2786)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2977..2998
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3161..3265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3266..3334,3335..4126)
                     /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(3484..3503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4297..4885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4786..4805
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5039..5056
                     /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 aggatccact cgaggcaggc agcacgctgg acaaaatcgc caaaaacggt
      241 gtgattgtcg tcggtcaccg tgaatcttca gtgcctttct cttattacga cgttgctggt
      301 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      361 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      421 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      481 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      541 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      601 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      661 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      721 aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa gcagaagaac
      781 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      841 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      901 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      961 ctgctggagt tcgtgaccgc cgccgggatc actctcggta gcaaccaaaa agtggtgggt
     1021 tactcgcagg attactccaa cgccattgtt gaagcagtga aaaagaaact caacaaaccg
     1081 gacttgcagg taaaactgat tccgattacc tcacaaaacc gtattccact gctgcaaaac
     1141 ggcactttcg attttgaatg tggttctacc accaacaacg tcgaacgcca aaaacaggcg
     1201 gctttctctg acactatttt cgtggtcggt acgcgcctgt tgaccaaaaa gggtggcgat
     1261 atcaaagatt ttgccaacct gaaagacaaa gccgtagtcg tcacttccgg cactacctct
     1321 gaagttttgc tcaacaaact gaatgaagag caaaaaatga atatgcgcat catcagcgcc
     1381 aaagatcacg gtgactcttt ccgcaccctg gaaagcggtc gtgccgttgc ctttatgatg
     1441 gatgactggc tgctggccgg tgaacgtgcg aaagcgaaga aaccagacaa ctgggaaatc
     1501 gtcggcaagc cgcagtctca ggaggcctac ggttgtatgt tgcgtaaaga tgatccgcag
     1561 ttcaaaaagc tgatggatga caccatcgct caggtgcaga cctccggtga agcggaaaaa
     1621 tggtttgata agtggttcaa aaatccaatt ccgccgaaaa acctgaacat gaatttcgaa
     1681 ctgtcagacg aaatgaaagc actgttcaaa gaaccgaatg acaaggcact gaacggtacc
     1741 ggtggaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1801 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1861 acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg
     1921 cccaccctcg tgaccaccct gggctacggc ctgcagtgct tcgcccgcta ccccgaccac
     1981 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2041 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2101 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2161 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcaccgc cgacaagcag
     2221 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag
     2281 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2341 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2401 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2461 aagtaaaagc ttgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2521 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     2581 ttgctgaaag gaggaactat atccggatct ggcgtaatag cgaagaggcc cgcaccgatc
     2641 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     2701 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2761 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2821 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2881 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2941 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3001 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3061 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3121 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3181 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3241 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3301 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3361 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3421 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3481 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3541 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3601 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3661 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3721 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3781 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3841 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3901 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3961 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4021 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4081 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4141 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4201 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4261 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4321 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4381 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4441 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4501 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4561 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4621 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4681 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4741 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4801 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4861 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4921 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4981 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5041 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     5101 ttggccgatt cattaatgca g
//
