LOCUS       sequence-70402-e        4217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bact exp vector encoding for the 6XHis tagged insensitive variant of
            the fluorescent redox probe roGFP with mut Cys147 to Serine, del 
            insertion147b and mutated Cys204 to Glut.Referred to as refGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4217)
  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 4217)
  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: refGFP1_pQE30
FEATURES             Location/Qualifiers
     source          1..4217
                     /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"
     primer_bind     complement(181..209)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     terminator      963..1057
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1101..1760
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1167..1186)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1825..1911
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1959..1981)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2067..2207
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2122..2141
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2222..2239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2393..2981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2473..2492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3152..3943,3944..4012))
                     /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     3775..3794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4013..4117)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4185..4203
                     /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 aaggacgacg ggaattacaa gacacgggcc
      481 gaagtgaagt ttgagggaga tactctggtg aacaggattg aactgaaagg gattgatttc
      541 aaggaggacg gaaacatcct gggccacaaa ctggaataca actacaacag tcataacgtg
      601 tacatcatgg ccgataagca gaaaaacggg attaaggcta acttcaagac ccggcacaac
      661 atcgaggacg gctccgtgca gctggcagat cattaccagc agaacacccc aattggcgac
      721 ggacccgtcc tgctgcctga taatcactat ctgagcacac agtccgccct gtctaaggac
      781 cccaatgaaa aaagagatca tatggtcctg ctggaatttg tcaccgccgc agggattact
      841 cacggaatgg atgaactgta taaggacgaa ctgtgataaa gatctggtac ctcgagattg
      901 ggatcggatc ccgggcccgt cgactgcaga ggcctgcatg caagcttaat tagctgagct
      961 tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac
     1021 gctcggttgc cgccgggcgt tttttattgg tgagaatcca agctagcttg gcgagatttt
     1081 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     1141 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     1201 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     1261 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaatttc
     1321 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg
     1381 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     1441 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt
     1501 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     1561 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     1621 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     1681 ccgtctgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     1741 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctgggg
     1801 taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1861 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccgctc
     1921 tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     1981 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     2041 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     2101 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     2161 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     2221 cctcgctcac tgactcgctg cgctcggtct gtcggctgcg gcgagcggta tcagctcact
     2281 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2341 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2401 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2461 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2521 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2581 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2641 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2701 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2761 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2821 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2881 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2941 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3001 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3061 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3121 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3181 tctcagcgat ctgtctattt cgttcatcca tagctgcctg actccccgtc gtgtagataa
     3241 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3301 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3361 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3421 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3481 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3541 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3601 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3661 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3721 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3781 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3841 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3901 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3961 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4021 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4081 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4141 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4201 ggccctttcg tcttcac
//
