LOCUS       sequence-70378-e        6253 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 roGFP with 
            Cysteine 147 to Serine mutation (roGFP C147S, C204).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  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 6253)
  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_C147S_pCDNA3
FEATURES             Location/Qualifiers
     source          1..6253
                     /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"
     primer_bind     1629..1649
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(1805..1823)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1806..1823)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1843..1860)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1849..2073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2119..2547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2206..2225)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2416..2437
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2556..2576)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2561..2891
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2742..2877
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2804..2823
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2958..3752
                     /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(3012..3031)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3622..3641
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3926..4047
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3963..3982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4017..4036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4096..4112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4096..4112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4109..4131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4120..4136
                     /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(4144..4150,4151..4168,4169..4174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4189..4210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4327..4344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4498..5086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4578..4597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5257..6048,6049..6117))
                     /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     5880..5899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6118..6222)
                     /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 gcttcaagga cgacgggaat tacaagacac gggccgaagt gaagtttgag
     1321 ggagatactc tggtgaacag gattgaactg aaagggattg atttcaagga ggacggaaac
     1381 atcctgggcc acaaactgga atacaactac aacagtcata acgtgtacat catggccgat
     1441 aagcagaaaa acgggattaa ggctaacttc aagacccggc acaacatcga ggacggctcc
     1501 gtgcagctgg cagatcatta ccagcagaac accccaattg gcgatggccc tgtcctttta
     1561 ccagacaacc attacctgtc cacatgctct gccctttcga aagatcccaa cgaaaagaga
     1621 gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa
     1681 ctatacaaag atgagctata aggtaccgag ctcggatcca ctagtaacgg ccgccagtgt
     1741 gctggaattc tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc
     1801 ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta
     1861 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1921 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1981 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2041 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2101 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2161 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2221 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc
     2281 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2341 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2401 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2461 tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc
     2521 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2581 aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2641 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2701 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     2761 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     2821 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     2881 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     2941 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3001 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3061 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3121 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3181 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3241 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3301 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3361 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3421 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3481 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3541 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3601 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3661 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3721 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     3781 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     3841 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3901 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3961 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4021 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4081 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4141 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4201 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4261 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4321 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4381 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4441 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4501 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4561 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4621 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4681 ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4741 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4801 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4861 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4921 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4981 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5041 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5101 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5161 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5221 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5281 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5341 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5401 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5461 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5521 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5581 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5641 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5701 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5761 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5821 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5881 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5941 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6001 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6061 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6121 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6181 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6241 gccacctgac gtc
//
