LOCUS       sequence-330737-        8164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optimized EKAREV FRET biosensor sensor for ERK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8164)
  AUTHORS   Ponsioen B, Post JB, Buissant des Amorie JR, Laskaris D, van Ineveld
            RL, Kersten S, Bertotti A, Sassi F, Sipieter F, Cappe B, Mertens S, 
            Verlaan-Klink I, Boj SF, Vries RGJ, Rehmann H, Vandenabeele P, 
            Riquet FB, Trusolino L, Bos JL, Snippert HJG
  TITLE     Quantifying single-cell ERK dynamics in colorectal cancer organoids 
            reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.
  JOURNAL   Nat Cell Biol. 2021 Apr;23(4):377-390. doi: 
            10.1038/s41556-021-00654-5. Epub 2021 Apr 1.
  PUBMED    33795873
REFERENCE   2  (bases 1 to 8164)
  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: 
            "10.1038/s41556-021-00654-5"; journalName: "Nat Cell Biol"; date: 
            "2021-04"; volume: "23"; issue: "4"; pages: "377-390"
COMMENT     Sequence Label: miniTol2 x EF1a_EKAREN5 + PGK-blast
FEATURES             Location/Qualifiers
     source          1..8164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(15..26,27..214)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        286..1448
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          517..1439
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1396..1416
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1554..1556,1557..1559,1560..2267)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     primer_bind     complement(1860..1879)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3456..3476
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3554..3688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3578..3597)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3632..3651
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3752..4251
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3787..3810)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4184..4203
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        4263..4310
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4329..4727
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4775..4792)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4781..5005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    join(5067..5229,5230..5241)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(5289..5307)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5290..5307)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5325..5341)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5325..5341)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5338..5360)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5349..5365
                     /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(5373..5379,5380..5397,5398..5403))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5418..5439
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5556..5573)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5727..6315)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5807..5826)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6486..7277,7278..7346))
                     /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     7109..7128
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7347..7451)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7529..7984)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7666..7687)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7878..7897
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8110..8132
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8124..8141
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8125..8141
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(8148..8164,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccagagg tgtaaagtac ttgagtaatt ttacttgatt actgtactta
       61 agtattattt ttggggattt ttactttact tgagtacaat taaaaatcaa tacttttact
      121 tttacttaat tacatttttt tagaaaaaaa agtacttttt actccttaca attttattta
      181 cagtcaaaaa gtacttattt tttggagatc acttcattct attttccctt gctattacca
      241 aaccaattga attgcgctga tgcccagttt aatttaaata gatctggctc cggtgcccgt
      301 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
      361 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
      421 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
      481 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc
      541 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctc
      601 cagtacgtga ttcttgatcc cgagctggag ccaggggcgg gccttgcgct ttaggagccc
      661 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
      721 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
      781 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaggatc
      841 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
      901 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
      961 ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct
     1021 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
     1081 gccctgctcc agggggctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
     1141 cacccacaca aaggaaaggg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
     1201 agtaccgggc gccgtccagg cacctcgatt agttctggag cttttggagt acgtcgtctt
     1261 taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
     1321 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     1381 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
     1441 tgtcgtgagg atcccccggg ctgcaggaat tcggcgcgcc acgcgtaccg atagcttcga
     1501 attctgcagt cgacgatatc actagtctcg agcattttgg caaagaattc ggcatggtga
     1561 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1621 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1681 tgaccctgaa gcttctatgc accaccggca agctgcccgt gccctggccc accctcgtga
     1741 ccaccctggg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     1801 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1861 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1921 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1981 agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag aacggcatca
     2041 aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact
     2101 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2161 gctaccagtc cgccctgttc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2221 agttcctgac cgccgccggg atcactgagg gcatgaacga gctgtacctc gagatggcgg
     2281 acgaggagaa gctgccgccc ggctgggaga agcgcatgag ccgcagctca ggccgagtgt
     2341 actacttcaa ccacatcact aacgccagcc agtgggagcg gcccagcggc aacagcagca
     2401 gtggtggcaa aaacgggcag ggggagcctg ccaggggtac cagtgctggt ggtagtgctg
     2461 gtggtagtgc tggtggttcc ggcagtgctg gtggtagtgc tggtggtagt accagtgctg
     2521 gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tagtgctggt ggttccggca
     2581 gtgctggtgg tagtgctggt ggtagtacca gtgctggtgg tagtgctggt ggtagtgctg
     2641 gtggtagtgc tggtggtagt gctggtggtt ccggaccaga tgtccctaga actccagtgg
     2701 atccagcatg gctgtcattc caatttccgg gcggccgcat ggtgagtaaa ggggaagaac
     2761 tgtttactgg cgtggtgcca attctggtcg aactggatgg agatgtaaat gggcataaat
     2821 ttagtgtgtc tggagaaggg gaaggagacg ctacatatgg gaaactgaca ctgaaattta
     2881 tttgtactac tggaaaactg cctgtgcctt ggcctacact cgtgacaacc ctgtcttggg
     2941 gagtgcagtg ttttgcccgg tatcctgatc atatgaaaca gcatgatttt ttcaaatctg
     3001 ccatgcctga gggatatgtc caggaacgga caattttctt taaagatgat gggaattata
     3061 aaacacgggc cgaagtgaaa tttgaagggg atacactggt gaatcggatt gaactgaaag
     3121 gaattgattt taaagaagat gggaatattc tgggccataa actggaatat aattatttca
     3181 gcgataatgt ctacattaca gccgataaac agaagaatgg gattaaagcc aattttaaaa
     3241 ttcggcataa tattgaagat ggaggggtgc agctcgccga tcattatcag cagaatacac
     3301 ctattgggga tgggcctgtg ctgctgcctg ataatcatta tctgagcaca cagtctgccc
     3361 tgagcaagga ccctaatgaa aagcgggacc atatggtcct gctggaattt ctgactgccg
     3421 ccggcattac actcgggatg gatgaactgt ctagacctaa gaagaagcgt aaggtgtaag
     3481 tcgacggtcg agtcgagacg ctagccaccg gtcgttaaca ttaagctaga actatagtga
     3541 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
     3601 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3661 aaccattata agctgcaata aacaagttaa caacgccggc catcgattaa actcaggttc
     3721 agggggaggt gtgggaggtt ttttaattct cgggtagggg aggcgctttt cccaaggcag
     3781 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     3841 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     3901 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     3961 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     4021 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     4081 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
     4141 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
     4201 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc
     4261 ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
     4321 actaaaccat ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg
     4381 ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta
     4441 gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag
     4501 aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg
     4561 cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc
     4621 tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag
     4681 ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaaccc gggagatggg
     4741 ggaggctaac tgagctctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     4801 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4861 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4921 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4981 gctggggatg cggtgggctc tatggcttct gaggttaatt aatttatgtt tcaggttcaa
     5041 gcttaaacaa gaatctctag ttttctttct tgcttttact tttacttcct taatactcaa
     5101 gtacaatttt aatggagtac ttttttactt ttactcaagt aagattctag ccagatactt
     5161 ttacttttaa ttgagtaaaa ttttccctaa gtacttgtac tttcacttga gtaaaatttt
     5221 tgagtacttt ttacacctct gctcgaccat atgggagagc taacgcattc attttatagc
     5281 ttgagtattc tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct
     5341 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5401 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5461 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5521 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5581 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5641 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5701 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5761 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5821 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5881 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5941 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6001 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6061 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6121 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6181 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6241 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6301 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6361 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6421 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6481 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6541 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6601 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6661 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6721 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6781 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6841 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6901 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6961 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7021 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7081 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7141 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7201 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7261 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7321 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7381 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7441 ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag
     7501 atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc
     7561 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     7621 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     7681 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     7741 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     7801 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     7861 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     7921 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     7981 gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     8041 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     8101 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac
     8161 tata
//
