LOCUS       sequence-171091-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent biosensor for super-resolution imaging of ERK activity; 
            targeted to plasma membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  AUTHORS   Mo GC, Ross B, Hertel F, Manna P, Yang X, Greenwald E, Booth C, 
            Plummer AM, Tenner B, Chen Z, Wang Y, Kennedy EJ, Cole PA, Fleming 
            KG, Palmer A, Jimenez R, Xiao J, Dedecker P, Zhang J
  TITLE     Genetically encoded biosensors for visualizing live-cell biochemical
            activity at super-resolution.
  JOURNAL   Nat Methods. 2017 Apr;14(4):427-434. doi: 10.1038/nmeth.4221. Epub 
            2017 Mar 13.
  PUBMED    28288122
REFERENCE   2  (bases 1 to 7410)
  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/nmeth.4221"; journalName: "Nat Methods"; date: "2017-04"; 
            volume: "14"; issue: "4"; pages: "427-434"
COMMENT     Sequence Label: Lyn-FLINC-EKAR1
FEATURES             Location/Qualifiers
     source          1..7410
                     /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     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             946..1617
                     /codon_start=1
                     /product="monomeric photoswitchable Dronpa-Green1 
                     fluorescent protein"
                     /label=Dronpa-Green1
                     /translation="MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK
                     EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT
                     NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDVNMALSL
                     EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ
                     AK"
     CDS             1792..2139
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             2206..2901
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     promoter        complement(2962..2980)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2963..2980)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3000..3017)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3006..3230
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3276..3704
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3363..3382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3573..3594
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3713..3733)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3718..4048
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3899..4034
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3961..3980
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4115..4909
                     /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(4169..4188)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4779..4798
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5083..5204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5120..5139)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5174..5193
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5253..5269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5253..5269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5266..5288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5277..5293
                     /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(5301..5307,5308..5325,5326..5331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5346..5367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5484..5501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5655..6243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5735..5754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6414..7205,7206..7274))
                     /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     7037..7056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7275..7379)
                     /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 gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg gctgcatcaa gagcaagcgc aaggacaagg atccgatgag tgtgattaaa
      961 ccagacatga agatcaagct gcgtatggaa ggcgctgtaa atggacaccc gttcgcgatt
     1021 gaaggagttg gccttgggaa gcctttcgag ggaaaacaga gtatggacct taaagtcaaa
     1081 gaaggcggac ctctgccttt cgcctatgac atcttgacaa ctgtgttctg ttacggcaac
     1141 agggtattcg ccaaataccc agaaaatata gtagactatt tcaagcagtc gtttcctgag
     1201 ggctactctt gggaacgaag catgaattac gaagacgggg gcatttgtaa cgcgacaaac
     1261 gacataaccc tggatggtga ctgttatatc tatgaaattc gatttgatgg tgtgaacttt
     1321 cctgccaatg gtccagttat gcagaagagg actgtgaaat gggagccatc cactgagaaa
     1381 ttgtatgtgc gtgatggagt gctgaagggt gatgttaaca tggctctgtc gcttgaagga
     1441 ggtggccatt accgatgtga cttcaaaact acttataaag ctaagaaggt tgtccagttg
     1501 ccagactatc actttgtgga ccaccacatt gagattaaaa gccacgacaa agattacagt
     1561 aatgttaatc tgcatgagca tgccgaagcg cattctgagc tgccgaggca ggccaagaga
     1621 tctatggcgg acgaggagaa gctgccgccc ggctgggaga agcgcatgag ccgcagctca
     1681 ggccgagtgt actacttcaa ccacatcact aacgccagcc agtgggagcg gcccagcggc
     1741 aacagcagca gtggtggcaa aaacgggcag ggggagcctg ccaggggtac cagtgctggt
     1801 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg ttccggcagt
     1861 gctggtggta gtgctggtgg tagtaccagt gctggtggta gtgctggtgg tagtgctggt
     1921 ggtagtgctg gtggtagtgc tggtggttcc ggcagtgctg gtggtagtgc tggtggtagt
     1981 accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt
     2041 ggttccggca gtgctggtgg tagtgctggt ggtagtacca gtgctggtgg tagtgctggt
     2101 ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggaccaga tgtccctaga
     2161 actccagtgg ataaagcaaa gctgtcattc caatttccgg gtaccatgag cgagctgatt
     2221 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     2281 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
     2341 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     2401 agcagaacct tcatcaacca cacccagggc atccccgatt tctttaagca gtccttccct
     2461 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     2521 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     2581 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc caacaccgag
     2641 atgctgtacc ccgctgacgg cggcctggaa ggcagaaccg acatggccct gaagctcgtg
     2701 ggcgggggcc acctgatctg caacttcaag accacataca gatccaagaa acccgctaag
     2761 aacctcaaga tgcccggcgt ctactatgtg gaccacagac tggaaagaat caaggaggcc
     2821 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     2881 agcaaactgg ggcacaagta agaattctgc agatatccat cacactggcg gccgctcgag
     2941 catgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     3001 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3061 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3121 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3181 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3241 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3301 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3361 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3421 tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3481 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3541 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3601 actcaaccct atctcggtct attcttttga tttataaggg attttgggga tttcggccta
     3661 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3721 tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta tgcaaagcat
     3781 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3841 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3901 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3961 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     4021 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     4081 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4141 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4201 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4261 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     4321 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4381 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4441 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4501 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4561 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4621 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4681 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4741 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4801 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4861 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4921 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4981 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5041 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5101 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5161 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5221 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5281 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5341 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5401 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5461 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5521 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5581 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5641 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5701 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5761 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5821 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     5881 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5941 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6001 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6061 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6121 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6181 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6301 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6361 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6421 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6481 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6541 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6601 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6661 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6721 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6781 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6841 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6901 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6961 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7021 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7081 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7141 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7201 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7261 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7321 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7381 acatttcccc gaaaagtgcc acctgacgtc
//
