LOCUS       sequence-360343-        5154 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 5154)
  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 5154)
  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-10u
FEATURES             Location/Qualifiers
     source          1..5154
                     /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             69..91
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             111..128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             132..164
                     /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(168..176,177..191)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(204..206,207..209,210..920)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1779..1781,1782..1784,1785..2498)
                     /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(2554..2572)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2568..2615
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2712..3167
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2799..2818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3009..3030
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3193..3297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3298..3366,3367..4158)
                     /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(3516..3535)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4329..4917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4818..4837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5071..5088
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
       61 gaaataattt tgtttaactt taagaaggag atatacatat gcggggttct catcatcatc
      121 atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg
      181 atgacgataa ggatccgggc cgcatggtga gcaagggcga ggagctgttc accggggtgg
      241 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      301 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      361 agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca
      421 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
      481 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
      541 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
      601 aggacggcaa catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata
      661 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
      721 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
      781 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
      841 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
      901 gcatggacga gctgtacaag ggtggtaccg gaggcgccgc aggcagcacg ctggacaaaa
      961 tcgccaaaaa cggtgtgatt gtcgtcggtc accgtgaatc ttcagtgcct ttctcttatt
     1021 acgacaatca gcaaaaagtg gtgggttact cgcaggatta ctccaacgcc attgttgaag
     1081 cagtgaaaaa gaaactcaac aaaccggact tgcaggtaaa actgattccg attacctcac
     1141 aaaaccgtat tccactgctg caaaacggca ctttcgattt tgaatgtggt tctaccacca
     1201 acaacgtcga acgccaaaaa caggcggctt tctctgacac tattttcgtg gtcggtacgc
     1261 gcctgttgac caaaaagggt ggcgatatca aagattttgc caacctgaaa gacaaagccg
     1321 tagtcgtcac ttccggcact acctctgaag ttttgctcaa caaactgaat gaagagcaaa
     1381 aaatgaatat gcgcatcatc agcgccaaag atcacggtga ctctttccgc accctggaaa
     1441 gcggtcgtgc cgttgccttt atgatggatg accggctgct ggccggtgaa cgtgcgaaag
     1501 cgaagaaacc agacaactgg gaaatcgtcg gcaagccgca gtctcaggag gcctacggtt
     1561 gtatgttgcg taaagatgat ccgcagttca aaaagctgat ggatgacacc atcgctcagg
     1621 tgcagacctc cggtgaagcg gaaaaatggt ttgataagtg gttcaaaaat ccaattccgc
     1681 cgaaaaacct gaacatgaat ttcgaactgt cagacgaaat gaaagcactg ttcaaagaac
     1741 cgaatgacaa ggcactgaac ggcgccggta ccggtggaat ggtgagcaag ggcgaggagc
     1801 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1861 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga
     1921 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg
     1981 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2041 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2101 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2161 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2221 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     2281 tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc
     2341 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc
     2401 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2461 ccgggatcac tctcggcatg gacgagctgt acaagtaaaa gcttgatccg gctgctaaca
     2521 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2581 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     2641 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2701 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2761 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2821 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2881 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgttggttc
     2941 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3001 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3061 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3121 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3181 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3241 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3301 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3361 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3421 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3481 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3541 attgacgccg ggcaagagca actcggtcgc cgcatacatt attctcagaa tgacttggtt
     3601 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3661 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3721 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3781 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3841 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3901 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3961 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4021 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4081 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4141 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4201 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4261 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4321 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4381 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4441 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4501 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4561 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4621 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4681 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4741 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4801 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4861 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4921 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4981 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5041 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5101 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagg
//
