LOCUS       sequence-70377-e        6194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding for the ER targeted and FLAGM1 
            tagged insensitive variant of fluorescent redox probe roGFPiE with 
            Cysteine 204 to Glutamine mutation (roGFP C147, E147b, C204Q).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6194)
  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 6194)
  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: ERroGFP_iE_C204Q_pCDNA3
FEATURES             Location/Qualifiers
     source          1..6194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     815..839
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             949..972
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1006..1034)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(1746..1764)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1747..1764)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1784..1801)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1790..2014
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2060..2488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2147..2166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2357..2378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2497..2517)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2502..2832
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2683..2818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2745..2764
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2899..3693
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2953..2972)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3563..3582
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3867..3988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3904..3923)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3958..3977
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4037..4053)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4037..4053)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4050..4072)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4061..4077
                     /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)."
     promoter        complement(join(4085..4091,4092..4109,4110..4115))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4130..4151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4268..4285)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4439..5027)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4519..4538)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5198..5989,5990..6058))
                     /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     5821..5840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6059..6163)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
      841 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgactc tagaggatcc
      901 gccatgagcg ccctgctgat cctggctctc gtgggcgctg ccgtggccga ctacaaggac
      961 gacgacgaca agtccggaag taaaggagaa gaacttttca ctggagttgt cccaattctt
     1021 gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt
     1081 gatgcaacat acggaaaact tacccttaaa tttatttcca ctactggaaa actacctgtt
     1141 ccatggccaa cacttgtcac tactctgagt tatggtgttc aatgcttttc aagataccca
     1201 gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa
     1261 agaactataa gcttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa
     1321 ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac
     1381 attcttggac acaaattgga atacaactat aactgcgagt ccaatgtata catcatggca
     1441 gacaaacaaa agaatggaat caaagcgaac ttcaaaacca gacacaacat tgaagatgga
     1501 agcgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgacgg acccgtcctg
     1561 ctgcctgata atcactatct gagcacacag tccgccctgt ctaaggaccc caatgaaaaa
     1621 agagatcata tggtcctgct ggaatttgtc accgccgcag ggattactca cggaatggat
     1681 gaactgtata aggacgaact gtgataaaga tctggtacct cgagcatgca tctagagggc
     1741 cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct
     1801 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1861 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     1921 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     1981 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2041 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2101 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcggggcatc
     2221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2401 gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt aaaaaatgag
     2461 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     2521 gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct caattagtca
     2581 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2641 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2701 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2761 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2821 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     2881 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2941 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3001 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3061 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3121 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3181 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3241 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3301 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3361 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3421 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3481 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3541 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3601 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3661 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3721 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3781 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3841 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3901 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3961 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4021 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4081 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4141 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4201 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4261 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4321 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4381 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4441 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4501 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4561 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4621 tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4681 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4741 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4801 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4861 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4921 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4981 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5041 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5101 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5161 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5221 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5281 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5341 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5401 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5461 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5521 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5581 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5641 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5701 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5761 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5821 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5881 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5941 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6001 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6061 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6121 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6181 tgccacctga cgtc
//
