LOCUS       sequence-7666-ex        5206 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 5206)
  AUTHORS   Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB
  TITLE     Detection of glutamate release from neurons by genetically encoded 
            surface-displayed FRET nanosensors.
  JOURNAL   Proc Natl Acad Sci U S A. 2005 Jun 14. 102(24):8740-5.
  PUBMED    15939876
REFERENCE   2  (bases 1 to 5206)
  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: "Proc Natl 
            Acad Sci U S A. 2005 Jun 14. 102(24):8740-5."
COMMENT     Sequence Label: pRSET FLIPE-1m
FEATURES             Location/Qualifiers
     source          1..5206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..38
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        19..37
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             67..89
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             109..126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             130..162
                     /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(166..174,175..189)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(202..204,205..207,208..918)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1777..1779,1780..1782,1783..2496)
                     /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(2552..2570)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2566..2613
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2710..3165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2797..2816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3007..3028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3246..3350
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3351..3419,3420..4211)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLID*MEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4382..4970
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4871..4890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5124..5141
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
       61 gataattttg tttaacttta agaaggagat atacatatgc ggggttctca tcatcatcat
      121 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat
      181 gacgataagg atccgggccg catggtgagc aagggcgagg agctgttcac cggggtggtg
      241 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      301 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      361 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc
      421 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      481 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      541 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      601 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc
      661 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
      721 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      781 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
      841 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      901 atggacgagc tgtacaaggg tctcgaggga ggcgccgcag gcagcacgct ggacaaaatc
      961 gccaaaaacg gtgtgattgt cgtcggtcac cgtgaatctt cagtgccttt ctcttattac
     1021 gacaatcagc aaaaagtggt gggttactcg caggattact ccaacgccat tgttgaagca
     1081 gtgaaaaaga aactcaacaa accggacttg caggtaaaac tgattccgat tacctcacaa
     1141 aaccgtattc cactgctgca aaacggcact ttcgattttg aatgtggttc taccaccaac
     1201 aacgtcgaac gccaaaaaca ggcggctttc tctgacacta ttttcgtggt cggtacgcgc
     1261 ctgttgacca aaaagggtgg cgatatcaaa gattttgcca acctgaaaga caaagccgta
     1321 gtcgtcactt ccggcactac ctctgaagtt ttgctcaaca aactgaatga agagcaaaaa
     1381 atgaatatgc gcatcatcag cgccaaagat cacggtgact ctttccgcac cctggaaagc
     1441 ggtcgtgccg ttgcctttat gatggatgac tggctgctgg ccggtgaacg tgcgaaagcg
     1501 aagaaaccag acaactggga aatcgtcggc aagccgcagt ctcaggaggc ctacggttgt
     1561 atgttgcgta aagatgatcc gcagttcaaa aagctgatgg atgacaccat cgctcaggtg
     1621 cagacctccg gtgaagcgga aaaatggttt gataagtggt tcaaaaatcc aattccgccg
     1681 aaaaacctga acatgaattt cgaactgtca gacgaaatga aagcactgtt caaagaaccg
     1741 aatgacaagg cactgaacgg cgccggtacc ggtggaatgg tgagcaaggg cgaggagctg
     1801 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1861 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc
     1921 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc
     1981 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2041 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2101 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2161 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2221 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     2281 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     2341 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg
     2401 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2461 gggatcactc tcggcatgga cgagctgtac aagtaaaagc ttgatccggc tgctaacaaa
     2521 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2581 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatct
     2641 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     2701 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2761 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2821 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2881 tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac
     2941 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3001 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatcgcg gtctattctt
     3061 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3121 aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat
     3181 tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa
     3241 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3301 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3361 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3421 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3481 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3541 ttccaatgat gagcactttt aaagttctgc tatgtgatac actattatcc cgtattgacg
     3601 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3661 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3721 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3781 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3841 aaccggagct gaatgaagcc ataccaaacg acgagagtga caccacgatg cctgtagcaa
     3901 tgccaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3961 aattaataga ctgaatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4021 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4081 ttgcagcact ggggccagat ggtaagcgct cccgtatcgt agttatctac acgacgggga
     4141 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4201 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4261 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4321 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4381 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4441 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4501 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4561 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4621 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4681 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4741 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4801 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4861 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4921 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4981 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg
     5041 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5101 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5161 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcag
//
