LOCUS       sequence-70381-e        4174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector encoding for the 6XHis tagged 
            insensitive variant of fluorescent redox probe roGFPiE with Cysteine
            204 to Serine mutation (roGFP C147S, E147b, C204).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4174)
  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 4174)
  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_C204S_pQE30
FEATURES             Location/Qualifiers
     source          1..4174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    30..46
                     /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    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             151..867
                     /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
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCESNVYIMADKQKNGIK
                     ANFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTSSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(181..209)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     807..827
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      920..1014
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1058..1717
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1124..1143)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1782..1868
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1916..1938)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2024..2164
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2079..2098
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2179..2196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2350..2938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2430..2449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3109..3900,3901..3969))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQAAMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3732..3751
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3970..4074)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4142..4160
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaatcac aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc atgagtaaag gagaagaact tttcactgga
      181 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
      241 ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttccactact
      301 ggaaaactac ctgttccatg gccaacactt gtcactactc tgagttatgg tgttcaatgc
      361 ttttcaagat acccagatca tatgaaacgg catgactttt tcaagagtgc catgcccgaa
      421 ggttatgtac aggaaagaac tataagcttc aaagatgacg ggaactacaa gacacgtgct
      481 gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt
      541 aaagaagatg gaaacattct tggacacaaa ttggaataca actataactg cgagtccaat
      601 gtatacatca tggcagacaa acaaaagaat ggaatcaaag cgaacttcaa aaccagacac
      661 aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc
      721 gatggccctg tccttttacc agacaaccat tacctgtcca catcctctgc cctttcgaaa
      781 gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt
      841 acacatggca tggatgaact atacaaataa taacgatccg agctcgagat ctgcagccaa
      901 gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat
      961 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc
     1021 tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     1081 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     1141 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     1201 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     1261 tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     1321 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     1381 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     1441 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     1501 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     1561 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1621 gattcaggtt catcatgccg tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1681 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
     1741 cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc
     1801 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     1861 ggacaaatcc gccgctctag agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     1921 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     1981 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     2041 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     2101 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     2161 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtctgtc ggctgcggcg
     2221 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2281 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2341 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2401 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2461 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2521 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     2581 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2641 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2701 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2761 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     2821 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2881 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2941 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3001 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3061 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3121 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ctgcctgact
     3181 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3241 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3301 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3361 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3421 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3481 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3541 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3601 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3661 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3721 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3781 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3841 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3901 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3961 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4021 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4081 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4141 aaataggcgt atcacgaggc cctttcgtct tcac
//
