LOCUS       sequence-70401-e        6191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mam vector encoding for the ER targeted and FLAGM1 tagged 
            insensitive variant of fluorescent redox probe roGFPiE with mut 
            Cys147 to Ser, del insertion147b and mut Cys204 to Glut.Referred to 
            as ref GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6191)
  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 6191)
  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: ERrefGFP1_pCDNA3
FEATURES             Location/Qualifiers
     source          1..6191
                     /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(1743..1761)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1744..1761)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1781..1798)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1787..2011
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2057..2485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2144..2163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2354..2375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2494..2514)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2499..2829
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2680..2815
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2742..2761
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2896..3690
                     /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(2950..2969)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3560..3579
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3864..3985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3901..3920)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3955..3974
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4034..4050)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4034..4050)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4047..4069)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4058..4074
                     /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(4082..4088,4089..4106,4107..4112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4127..4148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4265..4282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4436..5024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4516..4535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5195..5986,5987..6055))
                     /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     5818..5837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6056..6160)
                     /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 gcgacggacc cgtcctgctg
     1561 cctgataatc actatctgag cacacagtcc gccctgtcta aggaccccaa tgaaaaaaga
     1621 gatcatatgg tcctgctgga atttgtcacc gccgcaggga ttactcacgg aatggatgaa
     1681 ctgtataagg acgaactgtg ataaagatct ggtacctcga gcatgcatct agagggccct
     1741 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt
     1801 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1861 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1921 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1981 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2041 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2101 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2161 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct
     2221 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2281 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2341 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2401 tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg
     2461 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2521 agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2581 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2641 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2701 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2761 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2821 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     2881 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     2941 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3001 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3061 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3121 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3181 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3241 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3301 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3361 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3421 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3481 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3541 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3601 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3661 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3721 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3781 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3841 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     3901 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3961 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4021 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4081 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4141 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4201 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4261 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4321 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4441 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4621 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4861 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5041 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5101 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5161 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5221 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5281 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5341 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5401 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5461 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5521 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5581 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5641 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5701 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5761 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5821 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5881 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5941 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6001 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6061 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6121 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6181 cacctgacgt c
//
