LOCUS       sequence-409396-        7967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded FRET-based biosensor for monitoring ERK kinase 
            activity. Targeted to endosomes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7967)
  AUTHORS   Kwon Y, Mehta S, Clark M, Walters G, Zhong Y, Lee HN, Sunahara RK, 
            Zhang J
  TITLE     Non-canonical beta-adrenergic activation of ERK at endosomes.
  JOURNAL   Nature. 2022 Nov;611(7934):173-179. doi: 10.1038/s41586-022-05343-3.
            Epub 2022 Oct 26.
  PUBMED    36289326
REFERENCE   2  (bases 1 to 7967)
  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: 
            "10.1038/s41586-022-05343-3"; journalName: "Nature"; date: 
            "2022-11"; volume: "611"; issue: "7934"; pages: "173-179"
COMMENT     Sequence Label: pcDNA3-endo-EKAR4
FEATURES             Location/Qualifiers
     source          1..7967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(45..64)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        236..615
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        616..819
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     770..790
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     864..883
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        864..882
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      902..911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             923..940
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             944..976
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(980..988,989..1003)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1010..1012,1013..1015,1016..1528)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     primer_bind     1663..1684
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1871..2218
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     promoter        complement(3521..3539)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3522..3539)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3559..3576)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3565..3789
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3835..4263
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3922..3941)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4132..4153
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4272..4292)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4277..4606
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4457..4592
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4519..4538
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4673..5467
                     /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(4727..4746)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5337..5356
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5641..5762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5678..5697)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5732..5751
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5811..5827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5811..5827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5824..5846)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5835..5851
                     /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(5859..5865,5866..5883,5884..5889))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5904..5925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6042..6059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6213..6801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6293..6312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6972..7763,7764..7832))
                     /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     7595..7614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7833..7937)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
       61 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
      121 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
      181 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
      241 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      301 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      361 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      421 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      481 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      541 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      601 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      661 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      721 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      781 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc
      841 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agcttgcggc
      901 cgccaccatg gtgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
      961 tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatccca tggtgagcaa
     1021 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1081 cggccacagg ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1141 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1201 cctgacctgg ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1261 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgt accatcttct tcaaggacga
     1321 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1381 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1441 caactacatc agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     1501 ccacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     1561 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     1621 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1681 cgtgaccgcc gcccgcatgc acatggcgga cgaggagaag ctgccgcccg gctgggagaa
     1741 gcgcatgagc cgcagctcag gccgagtgta ctacttcaac cacatcacta acgccagcca
     1801 gtgggagcgg cccagcggca acagcagcag tggtggcaaa aacgggcagg gggagcctgc
     1861 caggggtacc agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag
     1921 tgctggtggt tccggcagtg ctggtggtag tgctggtggt agtaccagtg ctggtggtag
     1981 tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggttccg gcagtgctgg
     2041 tggtagtgct ggtggtagta ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag
     2101 tgctggtggt agtgctggtg gttccggcag tgctggtggt agtgctggtg gtagtaccag
     2161 tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc
     2221 cggaccagat gtccctagaa ctccagtgga taaagcaaag ctgtcattcc aattcccccg
     2281 cgcacgggag ctcatgtcta aaggtgaaga attattcacc ggcgtggtgc ccatcctggt
     2341 ggagctggac ggcgacgtga acggccacaa gttcagcgtg agcggcgagg gcgagggcga
     2401 cgccacctac ggcaagctga ccctgaagct gctgtgcacc accggcaagc tgcccgtgcc
     2461 ctggcccacc ctggtgacca ccctgggcta cggcgtgcag tgcttcgccc ggtaccccga
     2521 ccacatgaag cagcacgact tcttcaagag cgccatgccc gagggctacg tgcaggagcg
     2581 gaccatcttc ttcaaggacg acggcaacta caagacccgg gccgaggtga agttcgaggg
     2641 cgacaccctg gtgaaccgga tcgagctgaa gggcatcgac ttcaaggagg acagcaacat
     2701 cctgggccac aagctggagt acaactacaa cagccacaac gtgtacatca ccgccgacaa
     2761 gcagaagaac ggcatcaagg ccaacttcaa gatccggcac aacatcgagg acggcggcgt
     2821 gcagctggcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2881 cgataaccac tacctgagct accagagcgc cctgttcaag gaccccaacg agaagcggga
     2941 ccacatggtg ctgctggagt tcctgaccgc cgccggcatc accgagggta tgaatgaatt
     3001 gtacaaagaa ttccgaattg aattcgaaag tgatgccatg ttcgctgctg aaagagcccc
     3061 tgactgggtg gatgctgagg aatgccatcg gtgcagagta cagtttgggg tggtgacccg
     3121 caagcatcac tgccgagcat gtgggcagat cttctgtggc aagtgctcct ccaagtactc
     3181 caccatcccc aagttcggca ttgagaagga ggtgcgcgtg tgtgagccct gctatgagca
     3241 gctgaacaag aaggcacaag gacaaggatc cgaaagtgat gccatgttcg ctgctgaaag
     3301 agcccctgac tgggtggatg ctgaggaatg ccatcggtgc agagtacagt ttggggtggt
     3361 gacccgcaag catcactgcc gagcatgtgg gcagatcttc tgtggcaagt gctcctccaa
     3421 gtactccacc atccccaagt tcggcattga gaaggaggtg cgcgtgtgtg agccctgcta
     3481 tgagcagctg aacaagaagg cataatctag agggccctat tctatagtgt cacctaaatg
     3541 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3601 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3661 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3721 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3781 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3841 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3901 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3961 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4021 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     4081 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4141 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4201 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4261 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     4321 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4381 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4441 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4501 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4561 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     4621 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     4681 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     4741 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4801 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     4861 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4921 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4981 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     5041 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     5101 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     5161 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     5221 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     5281 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     5341 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     5401 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     5461 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     5521 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5581 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5641 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5701 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5761 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5821 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5881 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5941 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6001 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6061 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6121 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6181 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6241 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6301 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6361 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6421 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6481 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6541 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6601 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6661 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6721 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6781 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6841 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6901 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6961 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     7021 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     7081 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     7141 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     7201 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     7261 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     7321 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     7381 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7441 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7501 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7561 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7621 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7681 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7741 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7801 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7861 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7921 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgt
//
