LOCUS       sequence-469493-        6255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding for the ER targeted and FLAGM1 
            tagged redox probe roGFPiE (roGFP C147, E147b, C204) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6255)
  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 6255)
  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_pCDNA3
FEATURES             Location/Qualifiers
     source          1..6255
                     /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"
     CDS             979..1692
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ISTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCESNVYIMADKQKNGIKA
                     NFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1006..1034)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1632..1652
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     promoter        complement(1808..1826)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1809..1826)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1846..1863)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1852..2076
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2122..2550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2209..2228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2419..2440
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2559..2579)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2564..2893
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2744..2879
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2806..2825
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2960..3754
                     /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(3014..3033)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3624..3643
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3928..4049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3965..3984)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4019..4038
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4098..4114)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4098..4114)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4111..4133)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4122..4138
                     /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(4146..4152,4153..4170,4171..4176))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4191..4212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4329..4346)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4500..5088)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4580..4599)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5259..6050,6051..6119))
                     /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     5882..5901
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6120..6224)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 ttggcgatgg ccctgtcctt
     1561 ttaccagaca accattacct gtccacatgc tctgcccttt cgaaagatcc caacgaaaag
     1621 agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat
     1681 gaactataca aagatgagct ataaggtacc gagctcggat ccactagtaa cggccgccag
     1741 tgtgctggaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg
     1801 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     1861 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1921 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1981 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2041 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2101 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2161 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2221 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2281 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2341 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2401 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2461 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2521 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2581 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2641 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2701 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2761 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2821 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2881 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     2941 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3001 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3061 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3121 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3181 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3241 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3301 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3361 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3421 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3481 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3541 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3601 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3661 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3721 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     3781 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3841 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3901 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     3961 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4021 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4081 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4141 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4201 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4261 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4321 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4381 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4441 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4501 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4561 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4621 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4681 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4741 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4801 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4861 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4921 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4981 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5041 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5101 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5161 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5221 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5281 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5341 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5401 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5461 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5521 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5581 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5641 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5701 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5761 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5821 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5881 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5941 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6001 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6061 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6121 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6181 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6241 gtgccacctg acgtc
//
