LOCUS       sequence-426770-        4921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  in vitro MAPK-Sensor for luminescent detection of ERK and 
            p38-activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4921)
  AUTHORS   Poti AL, Denes L, Papp K, Bato C, Banoczi Z, Remenyi A, Alexa A
  TITLE     Phosphorylation-Assisted Luciferase Complementation Assay Designed 
            to Monitor Kinase Activity and Kinase-Domain-Mediated 
            Protein-Protein Binding.
  JOURNAL   Int J Mol Sci. 2023 Oct 3;24(19):14854. doi: 10.3390/ijms241914854.
  PUBMED    37834301
REFERENCE   2  (bases 1 to 4921)
  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: "Int J Mol 
            Sci."; date: "2023-10-3"; volume: "24(19):14854. doi"; pages: " 
            10.3390/ijms241914854"
COMMENT     Sequence Label: MEF2A-Sensor
FEATURES             Location/Qualifiers
     source          1..4921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        275..379
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(380..448,449..1240)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(598..617)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1411..1999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1900..1919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2153..2170
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2185..2324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2250..2269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2410..2432
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2426..2617)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3126..3145
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3126..3144
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3206..4306
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      4163..4172
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4280..4303
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4364..4384
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4547..4564
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4617..4635)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4631..4678
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(4715..4921,1..249)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4802..4821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
       61 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      121 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
      181 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
      241 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
      301 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      361 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      421 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      481 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      541 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
      601 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
      661 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
      721 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
      781 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
      841 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
      901 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
      961 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1021 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     1081 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1141 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1201 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1261 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1321 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1381 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1441 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1501 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     1561 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1621 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1681 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1741 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1801 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1861 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1921 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1981 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2041 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2101 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2161 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2221 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2281 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     2341 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     2401 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     2461 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     2521 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     2581 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     2641 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     2701 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     2761 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     2821 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     2881 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     2941 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     3001 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     3061 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatc tcgatcccgc
     3121 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagat tttcacgagc
     3181 acttcaccaa caaggaccat agattatgaa aactgaagaa ggtaaactgg taatctggat
     3241 taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac
     3301 cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc
     3361 ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc
     3421 tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc
     3481 gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga
     3541 agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga
     3601 gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct
     3661 gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta
     3721 tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg
     3781 tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc
     3841 catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc
     3901 atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa
     3961 gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc
     4021 gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga
     4081 agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt
     4141 ggcgaaagat ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg aaatcatgcc
     4201 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
     4261 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa
     4321 caacaacact agtggctccg gtggtggtgg cggttccatg agcgagaatc tttattttca
     4381 gggatccgct agccgcaaac ctgatctgcg tgttgttatt cctcctagcc atatgggcag
     4441 tggtagcagt ggcggtagca gcggctctgg tagcgtcgac gtacctcgta ccccggtgag
     4501 cggccgcgtg accggctacc ggctgttcga ggagattctg ctcgagcacc accaccacca
     4561 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     4621 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4681 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     4741 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4801 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4861 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4921 a
//
