LOCUS       sequence-363410-        7336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded FRET-based sensor for monitoring ERK activity 
            near a protein of interest. Must pair with GFP11-tagged POI to 
            reconstitute donor fluorescence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7336)
  AUTHORS   Tenner B, Zhang JZ, Kwon Y, Pessino V, Feng S, Huang B, Mehta S, 
            Zhang J
  TITLE     FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized 
            signaling within endogenous microdomains.
  JOURNAL   Sci Adv. 2021 May 21;7(21). pii: 7/21/eabe4091. doi: 
            10.1126/sciadv.abe4091. Print 2021 May.
  PUBMED    34020947
REFERENCE   2  (bases 1 to 7336)
  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"; journalName: "Sci Adv."; 
            date: "2021-05-21"; volume: "7(21). pii: 7/21/eabe4091. doi"; pages:
            " 10.1126/sciadv.abe4091. Print 2021 May"
COMMENT     Sequence Label: pcDNA3.1(+)-FluoSTEP-EKAR
FEATURES             Location/Qualifiers
     source          1..7336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     733..752
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        733..751
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             789..1499
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RITVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREVAV
                     AKFAGLGGGMDELYK"
     CDS             1677..2024
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             2100..2741
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     primer_bind     complement(2797..2814)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2803..3027
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3073..3501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3160..3179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3370..3391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3510..3530)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3515..3844
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3695..3830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3757..3776
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3911..4705
                     /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(3965..3984)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4575..4594
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4879..5000
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4916..4935)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4970..4989
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5049..5065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5049..5065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5062..5084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5073..5089
                     /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(5097..5103,5104..5121,5122..5127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5142..5163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5280..5297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5451..6039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5531..5550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6210..7001,7002..7070))
                     /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     6833..6852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7071..7175)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7250..7269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      721 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
      781 aagcttggat ggtgtctaag ggcgaagagc tgatcaagga aaatatgcgt atgaaggtgg
      841 tcatggaagg ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc
      901 cgtacatggg aactcaaacc atgaggatca cagtcatcga gggaggaccc ctgccatttg
      961 cctttgacat tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga
     1021 aaggcattcc tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta
     1081 cgagatacga agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct
     1141 gtctcgttta ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc
     1201 agaagaagac caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc
     1261 tgaggggata cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt
     1321 tcgtaacaac ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg
     1381 ccgttgatca ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac
     1441 gcgaagtcgc agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagc
     1501 gcatgcacat ggcggacgag gagaagctgc cgcccggctg ggagaagcgc atgagccgca
     1561 gctcaggccg agtgtactac ttcaaccaca tcactaacgc cagccagtgg gagcggccca
     1621 gcggcaacag cagcagtggt ggcaaaaacg ggcaggggga gcctgccagg ggtaccagtg
     1681 ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggttccg
     1741 gcagtgctgg tggtagtgct ggtggtagta ccagtgctgg tggtagtgct ggtggtagtg
     1801 ctggtggtag tgctggtggt agtgctggtg gttccggcag tgctggtggt agtgctggtg
     1861 gtagtaccag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg
     1921 ctggtggttc cggcagtgct ggtggtagtg ctggtggtag taccagtgct ggtggtagtg
     1981 ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggttccgga ccagatgtcc
     2041 ctagaactcc agtggataaa gcaaagctgt cattccaatt cccccgcgca cgggagctca
     2101 tgtccaaagg agaagaactg tttaccggtg ttgtgccaat tttggttgaa ctcgatggtg
     2161 atgtcaacgg acataagttc tcagtgagag gcgaaggaga aggtgacgcc accattggaa
     2221 aattgactct taaattcatc tgtactactg gtaaacttcc tgtaccatgg ccgactctcg
     2281 taacaacgct tacgtacgga gttcagtgct tttcgagata cccagaccat atgaaaagac
     2341 atgacttttt taagtcggct atgcctgaag gttacgtgca agaaagaaca atttcgttca
     2401 aagatgatgg aaaatataaa actagagcag ttgttaaatt tgaaggagat actttggtta
     2461 accgcattga actgaaagga acagatttta aagaagatgg taatattctt ggacacaaac
     2521 tcgaatacaa ttttaatagt cataacgtat acatcactgc tgataagcaa aagaacggaa
     2581 ttaaagcgaa tttcacagta cgccataatg tagaagatgg cagtgttcaa cttgccgacc
     2641 attaccaaca aaacacccct attggagacg gtccggtact tcttcctgat aatcactacc
     2701 tctcaacaca aacagtcctg agcaaagatc caaatgaaaa aggaacaggt ggcggcggaa
     2761 gttagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2821 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2881 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2941 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3001 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3061 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3121 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3181 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3241 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3301 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3361 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3421 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3481 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3541 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3601 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3661 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3721 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3781 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3841 caaaaagctc ccgggagctt atatatccat tttcggatct gatcaagaga caggatgagg
     3901 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3961 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4021 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4081 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4141 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4201 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4261 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4321 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4381 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4441 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4501 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4561 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4621 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4681 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4741 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4801 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4861 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4921 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4981 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5041 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5101 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5161 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5221 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5281 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5341 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5401 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5461 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5521 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5581 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5641 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5701 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5761 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5821 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5881 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5941 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6001 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6061 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6121 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6181 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6241 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6301 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6361 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6421 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6481 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6541 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6601 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6661 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6721 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6781 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6841 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6901 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6961 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7021 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7081 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7141 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7201 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     7261 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     7321 agtgcgcgag caaaat
//
