LOCUS       sequence-243128-        7496 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 7496)
  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 7496)
  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..7496
                     /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             3001..3021
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(3049..3067)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3050..3067)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3087..3104)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3093..3317
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3363..3791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3450..3469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3660..3681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3800..3820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3805..4134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3985..4120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4047..4066
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4201..4995
                     /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(4255..4274)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4865..4884
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5169..5290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5206..5225)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5260..5279
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5339..5355)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5339..5355)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5352..5374)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5363..5379
                     /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(5387..5393,5394..5411,5412..5417))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5432..5453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5570..5587)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5741..6329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5821..5840)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6500..7291,7292..7360))
                     /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     7123..7142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7361..7465)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 ccggcagcat gcacatggcg 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 ccaattcccc
     2281 cgcgcacggg agctcgcctc ctccgaggac aacaacatgg ccaccatcaa agagttcatg
     2341 cgcttcaagg tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     2401 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc
     2461 cccctgccct tcgcctggga catcctgtcc ccccagttca tgtacggctc cgaggcgtac
     2521 gtgaggcacc ccgccgacat ccccgattac aagaagctgc ccttccccga gggcttcaag
     2581 tgggagcgcg tgatgaactt cgaagacggc ggtctggtga ccgttaccca ggactcctcc
     2641 ctgcaggacg gcacgctgat ctgcaaggtg aagatgcgcg gcaccaactt cccccccgac
     2701 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagat gctgtacccc
     2761 gaagacggcg tgctgaaggg ccatagctat caggccctga agctgaagga cggcggccac
     2821 tacctggtgg agttcgagac catctacatg gccaagaagc ccgtgcaact gcccggcgat
     2881 tactgtgtgg acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa
     2941 cagtacgggc gctccgaggg ccgccaccgt ctgggcatgg acgagcggta caaggaattc
     3001 cccaaaaaga agagaaaggt ggaagacgca taatctagag ggccctattc tatagtgtca
     3061 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3121 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3181 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3241 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3301 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3361 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3421 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3481 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3541 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3601 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3661 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3721 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3781 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3841 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3901 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3961 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4021 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     4081 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4141 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4201 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4261 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4321 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4381 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4441 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4501 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4561 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4621 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4681 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4741 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4801 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4861 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4921 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4981 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5041 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5101 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5161 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5221 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5281 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5341 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5401 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5461 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5521 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6181 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6421 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6481 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6541 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6601 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6661 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6721 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6781 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6841 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6901 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6961 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7021 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7081 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7141 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7201 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7261 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7321 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7381 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7441 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
