LOCUS       sequence-182390-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLIM sensor for ERK activation in nucleus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  AUTHORS   Tang S, Yasuda R
  TITLE     Imaging ERK and PKA Activation in Single Dendritic Spines during 
            Structural Plasticity.
  JOURNAL   Neuron. 2017 Mar 22;93(6):1315-1324.e3. doi: 
            10.1016/j.neuron.2017.02.032. Epub 2017 Mar 9.
  PUBMED    28285819
REFERENCE   2  (bases 1 to 6028)
  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: 
            "10.1016/j.neuron.2017.02.032"; journalName: "Neuron"; date: 
            "2017-03-22- 22"; volume: "93"; issue: "6"; pages: "1315-1324.e3"
COMMENT     Sequence Label: EKARet-nuc
FEATURES             Location/Qualifiers
     source          1..6028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1058..1060,1061..1063,1064..1774)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITLGMDELYK"
     CDS             1949..2296
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGXGSAGGSAGGSXSAGGSAGGSXGGSAGGSAGGXGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             join(2363..2365,2366..2368,2369..3079)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2363..2365,2366..2368,2369..3079)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3086..3106
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3143..3264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3154..3173)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3208..3227
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3445..3900
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3532..3551)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3742..3763
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3967..3986)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4086..4108
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4146..4164)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4232..4336
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4337..4405,4406..5197)
                     /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     complement(4555..4574)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5368..5955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5856..5875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggcg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt acttaatacg actcactata ggctagcatg gtgagcaagg gcgaggagct
     1081 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1141 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctcat
     1201 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg
     1261 cctgatgtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1321 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1381 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1441 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actggaacag
     1501 cgtcaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1561 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1621 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct
     1681 gttcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcc tgaccgccgc
     1741 cgggatcact ctcggcatgg acgagctgta caagctcgag atggcggacg aggagaagct
     1801 gccgcccggc tgggagaagc gcatgagccg cagctcaggc cgagtgtact acttcaacca
     1861 catcactaac gccagccagt gggagcggcc cagcggcaac agcagcagtg gtggcaaaaa
     1921 cgggcagggg gagcctgcca ggggtaccag tgctggtggt agtgctggtg gtagtgctgg
     1981 tggtagtgct ggtggtagtg ctggtggttc cggcagtgct ggtggtagtg ctggtggtag
     2041 taccagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg
     2101 tggttscggc agtgctggtg gtagtgctgg tggtagtrcc agtgctggtg gtagtgctgg
     2161 tggtagtgst ggtggtagtg ctggtggtag tgctggtggt tscggcagtg ctggtggtag
     2221 tgctggtggt agtaccagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg
     2281 tggtagtgct ggtggttccg gaccagatgt ccctagaact ccagtggata aagcaaagct
     2341 gtcattccaa tttccgacgc gtatggtgag caagggcgag gagctgttca ccggggtggt
     2401 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2461 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2521 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     2581 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2641 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2701 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2761 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2821 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2881 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2941 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3001 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3061 catggacgag ctgtacaagt ctagacctaa gaagaagcgt aaggtgtaaa ctagtatctg
     3121 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3181 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3241 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3301 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3361 gataaggatc cgggctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3421 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3481 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3541 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3601 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3661 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3721 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3781 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3841 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3901 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     3961 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4021 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4081 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4141 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4201 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4261 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4321 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4381 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4441 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4501 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag tgctgctatg
     4561 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4621 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4681 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4741 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4801 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4861 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4921 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4981 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5041 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5101 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5161 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5221 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5281 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5341 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5401 cttgcaaaca aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6001 ccttttgctc acatggctcg acagatct
//
