LOCUS       sequence-201709-        7594 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express ERK-SPARK, an activity reporter of ERK in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7594)
  AUTHORS   Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis 
            A, Coughlin SR, Kornberg TB, Shu X
  TITLE     Visualizing Dynamics of Cell Signaling In Vivo with a Phase 
            Separation-Based Kinase Reporter.
  JOURNAL   Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008.
  PUBMED    29307513
REFERENCE   2  (bases 1 to 7594)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008."
COMMENT     Sequence Label: pcDNA3-ERK-SPARK
FEATURES             Location/Qualifiers
     source          1..7594
                     /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             join(1057..1059,1060..1062,1063..1773)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1969..2022
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      2029..2038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2239..2241,2242..2244,2245..2955)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3188..3205)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3194..3418
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3464..3892
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3551..3570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3761..3782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3901..3921)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3906..4235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4086..4221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4148..4167
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4302..5096
                     /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(4356..4375)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4966..4985
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5270..5391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5307..5326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5361..5380
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5440..5456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5440..5456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5453..5475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5464..5480
                     /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(5488..5494,5495..5512,5513..5518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5533..5554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5671..5688)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5842..6427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5922..5941)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6598..7389,7390..7458))
                     /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     7221..7240
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7459..7563)
                     /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 gctggctagc
      901 gccaccatgg ctgaccaact gactgaagag tggtcgacgg taccgcggac cggtcctgac
      961 gtaccacgta caccagtagg aaaggcaaag ctgtcattcc aattcccggg cggactcgag
     1021 tccggactca gatctggcag cggtggaggc gatatcatgg tgagcaaggg cgaggagctg
     1081 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1141 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1201 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1261 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1321 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1381 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1441 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1501 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     1561 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1621 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1681 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1741 gggatcactc tcggcatgga cgagctgtac aagggatccg gcagtgctgg tggtagtgct
     1801 ggtggtagtg ctggtggtag tgctggtggc agtgctggtg gtagtgctgg tggtagtgct
     1861 ggtggctcta gaggagaaat cgcaaagagt ctgaaagaaa ttgcaaaatc cttgaaggaa
     1921 atagcgtggt cactgaaaga aattgccaag tccctcaaag ggggtaccga gggcagagga
     1981 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caaagcttgc caccatggcg
     2041 gacgaggaga agctgccgcc cggctgggag aagcgcatga gccgcagctc aggccgagtg
     2101 tactacttca accacatcac taacgccagc cagtgggagc ggcccagcgg caacagcagc
     2161 agtggtggca aaaacgggca gggggagcct gccaggctcg agtccggact cagatctggc
     2221 agcggtggag gcgatatcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2281 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2341 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2401 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2461 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2521 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2581 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2641 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2701 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     2761 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2821 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2881 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2941 gacgagctgt acaagggatc cggcagtgct ggtggtagtg ctggtggtag tgctggtggt
     3001 agtgctggtg gcagtgctgg tggtagtgct ggtggtagtg ctggtggctc tagaaccttg
     3061 agagaaatcg aggaattgtt gaggaaaatc attgaagaca gcgtgcgcag tgtggccgag
     3121 ctggaggaca ttgagaagtg gctgaagaaa atctaagaat tcgggcccgt ttaaacccgc
     3181 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3241 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3301 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3361 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3421 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3481 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3541 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3601 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3661 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3721 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3781 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3841 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3901 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3961 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4021 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4081 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4141 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     4201 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4261 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4321 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4381 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4441 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     4501 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4561 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4621 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4681 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4741 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4801 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4861 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4921 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4981 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     5041 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     5101 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     5161 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5221 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     5281 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5341 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5401 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5461 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5521 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5581 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5641 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5701 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5761 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5821 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5881 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5941 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6001 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6061 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6121 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6181 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6241 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6301 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6361 aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6421 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6481 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6541 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6601 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6661 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6721 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6781 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6841 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6901 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6961 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7021 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7081 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7141 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7201 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7261 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7321 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7381 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7441 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7501 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7561 gcgcacattt ccccgaaaag tgccacctga cgtc
//
