LOCUS       sequence-330739-        8151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  control sensor for EKAREN5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8151)
  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 8151)
  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(TA) + PGK-blast
FEATURES             Location/Qualifiers
     source          1..8151
                     /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(1556..1558,1559..1561,1562..2269)
                     /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(1862..1881)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3458..3478
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3556..3690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3580..3599)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3634..3653
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3754..4253
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3789..3812)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4186..4205
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        4265..4312
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4331..4729
                     /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(4777..4794)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4783..5007
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    join(5051..5213,5214..5225)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(5276..5294)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5277..5294)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5312..5328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5312..5328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5325..5347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5336..5352
                     /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(5360..5366,5367..5384,5385..5390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5405..5426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5543..5560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5714..6302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5794..5813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6473..7264,7265..7333))
                     /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     7096..7115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7334..7438)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7516..7971)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7653..7674)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7865..7884
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8097..8119
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8111..8128
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8112..8128
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(8135..8151,1..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 atcaaacccg ggctgcagga attcggcgcg ccacgcgtac cgatagcttc
     1501 gaattctgca gtcgacgata tcactagtct cgagcatttt ggcaaagaat tcggcatggt
     1561 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1621 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1681 gctgaccctg aagcttctat gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1741 gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     1801 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1861 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1921 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1981 ggagtacaac tacaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
     2041 caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
     2101 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2161 gagctaccag tccgccctgt tcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2221 ggagttcctg accgccgccg ggatcactga gggcatgaac gagctgtacc tcgagatggc
     2281 ggacgaggag aagctgccgc ccggctggga gaagcgcatg agccgcagct caggccgagt
     2341 gtactacttc aaccacatca ctaacgccag ccagtgggag cggcccagcg gcaacagcag
     2401 cagtggtggc aaaaacgggc agggggagcc tgccaggggt accagtgctg gtggtagtgc
     2461 tggtggtagt gctggtggtt ccggcagtgc tggtggtagt gctggtggta gtaccagtgc
     2521 tggtggtagt gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggttccgg
     2581 cagtgctggt ggtagtgctg gtggtagtac cagtgctggt ggtagtgctg gtggtagtgc
     2641 tggtggtagt gctggtggta gtgctggtgg ttccggacca gatgtcccta gagctccagt
     2701 ggatccagca tggctgtcat tccaatttcc gggcggccgc atggtgagta aaggggaaga
     2761 actgtttact ggcgtggtgc caattctggt cgaactggat ggagatgtaa atgggcataa
     2821 atttagtgtg tctggagaag gggaaggaga cgctacatat gggaaactga cactgaaatt
     2881 tatttgtact actggaaaac tgcctgtgcc ttggcctaca ctcgtgacaa ccctgtcttg
     2941 gggagtgcag tgttttgccc ggtatcctga tcatatgaaa cagcatgatt ttttcaaatc
     3001 tgccatgcct gagggatatg tccaggaacg gacaattttc tttaaagatg atgggaatta
     3061 taaaacacgg gccgaagtga aatttgaagg ggatacactg gtgaatcgga ttgaactgaa
     3121 aggaattgat tttaaagaag atgggaatat tctgggccat aaactggaat ataattattt
     3181 cagcgataat gtctacatta cagccgataa acagaagaat gggattaaag ccaattttaa
     3241 aattcggcat aatattgaag atggaggggt gcagctcgcc gatcattatc agcagaatac
     3301 acctattggg gatgggcctg tgctgctgcc tgataatcat tatctgagca cacagtctgc
     3361 cctgagcaag gaccctaatg aaaagcggga ccatatggtc ctgctggaat ttctgactgc
     3421 cgccggcatt acactcggga tggatgaact gtctagacct aagaagaagc gtaaggtgta
     3481 agtcgacggt cgagtcgaga cgctagccac cggtcgttaa cattaagcta gaactatagt
     3541 gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca
     3601 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3661 gtaaccatta taagctgcaa taaacaagtt aacaacgccg gccatcgatt aaactcaggt
     3721 tcagggggag gtgtgggagg ttttttaatt ctcgggtagg ggaggcgctt ttcccaaggc
     3781 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     3841 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     3901 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     3961 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     4021 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     4081 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     4141 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     4201 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     4261 gcctgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     4321 gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     4381 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     4441 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     4501 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     4561 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     4621 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     4681 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac ccgggagatg
     4741 ggggaggcta actgagctct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     4801 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4861 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4921 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4981 atgctgggga tgcggtgggc tctatggctt ctgaggttaa ttaagcttaa acaagaatct
     5041 ctagttttct ttcttgcttt tacttttact tccttaatac tcaagtacaa ttttaatgga
     5101 gtactttttt acttttactc aagtaagatt ctagccagat acttttactt ttaattgagt
     5161 aaaattttcc ctaagtactt gtactttcac ttgagtaaaa tttttgagta ctttttacac
     5221 ctctgctcga ccatatggga gagctcccaa cgcgttggat gcatagcttg agtattctat
     5281 agtgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5341 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5401 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5461 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5521 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5581 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5641 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5701 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5761 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5941 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6001 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6061 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6121 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6181 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6241 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6301 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6361 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6421 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6481 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6541 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6601 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6661 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6721 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6781 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6841 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6901 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6961 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7021 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7081 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7141 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7201 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7261 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7321 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7381 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7441 catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     7501 aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     7561 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     7621 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     7681 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     7741 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     7801 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     7861 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     7921 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc
     7981 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     8041 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     8101 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat a
//
