LOCUS       sequence-72296-e        1126 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Bacterial expression vector encoding for the 6XHis tagged 
            insensitive variant of fluorescent redox probe roGFPiE with Cysteine
            147 to Serine mutation (roGFP C147S, E147b, C204).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1126)
  AUTHORS   Avezov E, Cross BC, Kaminski Schierle GS, Winters M, Harding HP, 
            Melo EP, Kaminski CF, Ron D
  TITLE     Lifetime imaging of a fluorescent protein sensor reveals surprising 
            stability of ER thiol redox.
  JOURNAL   J Cell Biol. 2013 Apr 15;201(2):337-49. doi: 10.1083/jcb.201211155.
  PUBMED    23589496
REFERENCE   2  (bases 1 to 1126)
  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"; doi: "10.1083/jcb"; 
            journalName: "J Cell Biol"; date: "2013-04-15- 15"; volume: "201"; 
            issue: "2"; pages: "337-49"
COMMENT     Sequence Label: roGFP_iE_C147S_pQE30
FEATURES             Location/Qualifiers
     source          1..1126
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     27..45
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    89..105
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    121..137
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             156..167
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             186..203
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             210..926
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FISTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSESNVYIMADKQKNGIK
                     ANFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(240..268)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     866..886
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      979..1073
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcacc
       61 tcgagaaatc ataaaaaatt tatttgcttt gtgagcggat aacaatcaca atagattcaa
      121 ttgtgagcgg ataacaattt cacacagaat tcattaaaga ggagaaatta actatgagag
      181 gatcgcatca ccatcaccat cacggatcca tgagtaaagg agaagaactt ttcactggag
      241 ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg
      301 gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tccactactg
      361 gaaaactacc tgttccatgg ccaacacttg tcactactct gagttatggt gttcaatgct
      421 tttcaagata cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag
      481 gttatgtaca ggaaagaact ataagcttca aagatgacgg gaactacaag acacgtgctg
      541 aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta
      601 aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactcc gagtccaatg
      661 tatacatcat ggcagacaaa caaaagaatg gaatcaaagc gaacttcaaa accagacaca
      721 acattgaaga tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg
      781 atggccctgt ccttttacca gacaaccatt acctgtccac atgctctgcc ctttcgaaag
      841 atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta
      901 cacatggcat ggatgaacta tacaaataat aacgatccga gctcgagatc tgcagccaag
      961 cttaattagc tgagcttgga ctcctgttga tagatccagt aatgacctca gaactccatc
     1021 tggatttgtt cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagct
     1081 agcttggcga gattttcagg agctaaggaa gctaaaatgg agaaaa
//
