LOCUS       Exported                6256 bp DNA     circular SYN 27-MAR-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 6256)
  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 6256)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6256)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6256)
  AUTHORS   .
  TITLE     Direct Submission
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     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6256
                     /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     815..839
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             904..948
                     /codon_start=1
                     /label=signal peptide
                     /translation="MSALLILALVGAAVA"
     CDS             949..972
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             979..1689
                     /codon_start=1
                     /label=roGFPiE
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ISTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCESNVYIMADKQKNGIKA
                     NFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTCSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELY"
     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"
     polyA_signal    1852..2076
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2122..2550
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2564..2894
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2961..3752
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3929..4062
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4099..4115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4123..4139)
                     /label=lac operator
                     /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(4147..4177)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4192..4213)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4501..5089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5263..6120)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6121..6225)
                     /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 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 aatcggggca
     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 tggggatttc ggcctattgg ttaaaaaatg
     2521 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2581 tggaaagtcc ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt
     2641 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2701 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2761 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2821 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2881 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     2941 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3001 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3061 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3121 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3181 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3241 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3301 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3361 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3421 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3481 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     3541 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     3601 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     3661 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     3721 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     3781 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     3841 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     3901 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     3961 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4021 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4081 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4261 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4681 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4921 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6241 agtgccacct gacgtc
//
