LOCUS       sequence-223859-        7488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ddRFP-based single-color ERK biosensor targeted to the nucleus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7488)
  AUTHORS   Mehta S, Zhang Y, Roth RH, Zhang JF, Mo A, Tenner B, Huganir RL, 
            Zhang J
  TITLE     Single-fluorophore biosensors for sensitive and multiplexed 
            detection of signalling activities.
  JOURNAL   Nat Cell Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6.
  PUBMED    30250062
REFERENCE   2  (bases 1 to 7488)
  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: "Nat Cell 
            Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi: 
            10.1038/s41556-018-0200-6."
COMMENT     Sequence Label: pcDNA3-RAB-EKARev-NLS
FEATURES             Location/Qualifiers
     source          1..7488
                     /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             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1873..2220
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             2992..3012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3040..3058)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3041..3058)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3078..3095)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3084..3308
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3354..3782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3441..3460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3651..3672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3791..3811)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3796..4126
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3977..4112
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4039..4058
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4193..4987
                     /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(4247..4266)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4857..4876
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5161..5282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5198..5217)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5252..5271
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5331..5347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5331..5347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5344..5366)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5355..5371
                     /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(5379..5385,5386..5403,5404..5409))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5424..5445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5562..5579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5733..6321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5813..5832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6492..7283,7284..7352))
                     /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     7115..7134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7353..7457)
                     /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 tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggtgagcaag
     1021 agcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc gcttggaggg ctccatgaac
     1081 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     1141 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     1201 cagatcatgt acggctccaa ggcgtacgtg aagcaccccg ccgacgtccc cgattacatg
     1261 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgcacttcga ggacggcggt
     1321 ctggtgaccg tgactcagga cacctccctg caggacggca cgctgatcta caaggtgaag
     1381 atgcgcggca ccaacttccc ccccgacggc cccgtaatgc agaagaagac cttgggctgg
     1441 gattatgcca ccgagcgcct gtaccccgaa gacggcgtgc tgaagggcga gcttctgggg
     1501 cgcctgaagc tgaaggacgg cggcctcaac ctggtggagt tcaagaccat ctacatggcc
     1561 aagaagcccg tgcaactgcc cggctactac ttcgtggaca ccaagctggg catcacctcc
     1621 cacaacgagg actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg
     1681 gggcatggca ccggcagcag atctatggcg gacgaggaga agctgccgcc cggctgggag
     1741 aagcgcatga gccgcagctc aggccgagtg tactacttca accacatcac taacgccagc
     1801 cagtgggagc ggcccagcgg caacagcagc agtggtggca aaaacgggca gggggagcct
     1861 gccaggggta ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt
     1921 agtgctggtg gttccggcag tgctggtggt agtgctggtg gtagtaccag tgctggtggt
     1981 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc cggcagtgct
     2041 ggtggtagtg ctggtggtag taccagtgct ggtggtagtg ctggtggtag tgctggtggt
     2101 agtgctggtg gtagtgctgg tggttccggc agtgctggtg gtagtgctgg tggtagtacc
     2161 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt
     2221 tccggaccag atgtccctag aactccagtg gataaagcaa agctgtcatt ccaatttccg
     2281 ggtaccgcct cctccgagga caacaacatg gccaccatca aagagttcat gcgcttcaag
     2341 gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
     2401 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
     2461 ttcgcctggg acatcctgtc cccccagttc atgtacggct ccgaggcgta cgtgaggcac
     2521 cccgccgaca tccccgatta caagaagctg cccttccccg agggcttcaa gtgggagcgc
     2581 gtgatgaact tcgaagacgg cggtctggtg accgttaccc aggactcctc cctgcaggac
     2641 ggcacgctga tctgcaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta
     2701 atgcagaaga agaccatggg ctgggaggcc tccaccgaga tgctgtaccc cgaagacggc
     2761 gtgctgaagg gccatagcta tcaggccctg aagctgaagg acggcggcca ctacctggtg
     2821 gagttcgaga ccatctacat ggccaagaag cccgtgcaac tgcccggcga ttactgtgtg
     2881 gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacggg
     2941 cgctccgagg gccgccaccg tctgggcatg gacgagcggt acaaggaatt ccccaaaaag
     3001 aagagaaagg tggaagacgc ataatctaga gggccctatt ctatagtgtc acctaaatgc
     3061 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3121 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3181 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3241 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3301 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3361 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3421 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3481 cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt
     3541 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3601 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3661 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3721 tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3781 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccaggcagg
     3841 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3901 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3961 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4021 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4081 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     4141 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     4201 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     4261 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4321 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     4381 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4441 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4501 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4561 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4621 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4681 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4741 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4801 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4861 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4921 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4981 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     5041 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5101 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5161 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5221 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5281 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5341 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5401 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5461 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5521 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5581 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5641 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5701 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5761 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5821 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5881 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt
     5941 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6001 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6061 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6121 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     6181 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6241 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6301 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6361 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6421 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6481 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6541 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6601 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6661 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6721 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6781 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6841 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6901 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6961 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7021 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7081 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7141 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7201 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7261 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7321 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7381 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7441 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
