LOCUS       sequence-417426-        7645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for in cell monitoring of receptor tyrosine 
            kinase-MAP ERK pathway activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  AUTHORS   Gudernova I, Foldynova-Trantirkova S, Ghannamova BE, Fafilek B, 
            Varecha M, Balek L, Hruba E, Jonatova L, Jelinkova I, Kunova 
            Bosakova M, Trantirek L, Mayer J, Krejci P
  TITLE     One reporter for in-cell activity profiling of majority of protein 
            kinase oncogenes.
  JOURNAL   Elife. 2017 Feb 15;6. pii: e21536. doi: 10.7554/eLife.21536.
  PUBMED    28199182
REFERENCE   2  (bases 1 to 7645)
  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: "Elife."; 
            date: "2017-02-15"; volume: "6. pii: e21536. doi"; pages: " 
            10.7554/eLife.21536"
COMMENT     Sequence Label: pKrox24(2xD-E)dTomato
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      2021..2030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2027..3457
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    3466..3514
                     /note="synthetic polyadenylation signal"
     rep_origin      3648..4076
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3735..3754)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3945..3966
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4085..4105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4090..4419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4270..4405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4332..4351
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4486..5280
                     /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(4540..4559)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5150..5169
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5454..5575
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5491..5510)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5545..5564
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5624..5640)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5624..5640)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5637..5659)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5648..5664
                     /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(5672..5678,5679..5696,5697..5702))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5717..5738
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5855..5872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6026..6614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6106..6125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6785..7576,7577..7645))
                     /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     7408..7427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtcgacg gatcgggaga tcttggaatt cggcctaact ggccggtacc
      181 aaagacaccg tgccatagat cgaggcccgg ggtcaaggcc ccgcctctcc tgggcggccc
      241 ctgcccaggc gggcccagcc gctcctcccc cgcactcccg gttcgctctc acggtccctg
      301 aggtgggcgg gcgggccctg gatgacagcg atagaacccc ggcccgactc gccctcgccc
      361 ccgctctggg tctgggcttc cccagcctag ttcacgccta ggagccgcct gagcagccgc
      421 gcgcccagcg ccacacgcca cgagccctcc ccgcctgggc gtccccggat cccgcgagcg
      481 ctcgggctcc cggcttggaa ccagggagga gggagggagc gagggagcaa ccagctgcga
      541 cccggaaatg ccatataagg agcaggaagg atcccccgcc ggaaagacac cgtgccatag
      601 atcgaggccc ggggtcaagg ccccgcctct cctgggcggc ccctgcccag gcgggcccag
      661 ccgctcctcc cccgcactcc cggttcgctc tcacggtccc tgaggtgggc gggcgggccc
      721 tggatgacag cgatagaacc ccggcccgac tcgccctcgc ccccgctctg ggtctgggct
      781 tccccagcct agttcacgcc taggagccgc ctgagcagcc gcgcgcccag cgccacacgc
      841 cacgagccct ccccgcctgg gcgtccccgg atcccgcgag cgctcgggct cccggcttgg
      901 aaccagggag gagggaggga gcgagggagc aaccagctgc gacccggaaa tgccatataa
      961 ggagcaggaa ggatcccccg ccgggagctc ggtaccaaag acaccgtgcc atagatcgag
     1021 gcccggggtc aaggccccgc ctctcctggg cggcccctgc ccaggcgggc ccagccgctc
     1081 ctcccccgca ctcccggttc gctctcacgg tccctgaggt gggcgggcgg gccctgggtg
     1141 acagcgatag aaccccggcc cgactcgccc tcgcccccgc tctgggtctg ggcttcccca
     1201 gcctagttca cgcctaggag ccgcctgagc agccgcgcgc ccagcgccac acgccacgag
     1261 ccctccccgc ctgggcgtcc ccggatcccg cgagcgctcg ggctcccggc ttggaaccag
     1321 ggaggaggga gggagcgagg gagcaaccag ctgcgacccg gaaatgccat ataaggagca
     1381 ggaaggatcc cccgccggaa caacccttat ttgggcagca ccttatttgg agtggcccga
     1441 tatggcccgg ccgcttccgg ctctgggagg agggaagaag gcggagggag gggcaacgcg
     1501 ggaactccgg agctgcgcgg gtcccggagg ccccggcggc ggctagagct ctaggcttcc
     1561 ccgaagcctg ggcgcctggg atgcgggcgc gggcgcgggc cctagggtgc aggatggagg
     1621 tgccgggcgc tgtcggatgg ggggcttcac gtcactccgg gtcctcccgg ccggtcctgc
     1681 catattaggg cttcctgctt cccatatatg gccatgtacg tcacgacgga ggcggacccg
     1741 tgccgttcca gacccttcaa atagaggcgg atccggggag tcgcgagaga tcccagcgcg
     1801 cagaacttgg ggagccgccg ccgccatccg ccgccgcagc cagcttccgc cgccgcagga
     1861 ccggcccctg ccccagcctc cgcagccgcg gcgcgtccac gcccgcccgc gcccagggcg
     1921 agtcggggtc gccgcctgca cgcttctcag tgttccgata tcctcgagcc caagcttgca
     1981 tgcctgcagg tcgactctag aggatccccg ggtaccggtc gccaccatgg tgagcaaggg
     2041 cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct ccatgaacgg
     2101 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     2161 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
     2221 gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg attacaagaa
     2281 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggtct
     2341 ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca aggtgaagat
     2401 gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca tgggctggga
     2461 ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccaccaggc
     2521 cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct acatggccaa
     2581 gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca tcacctccca
     2641 caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc accacctgtt
     2701 cctggggcat ggcaccggca gcaccggcag cggcagctcc ggcaccgcct cctccgagga
     2761 caacaacatg gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat
     2821 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     2881 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc
     2941 cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta
     3001 caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     3061 cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt
     3121 gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg
     3181 ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca
     3241 ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat
     3301 ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac
     3361 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca
     3421 cctgttcctg tacggcatgg acgagctgta caagtaggcg gccgcaataa aatatcttta
     3481 ttttcattac atctgtgtgt tggttttttg tgtgtctaga aataattctt actgtcatgc
     3541 caagtaagat gcttttctgt gctgcaatag caggcatgct ggggatgcgg tgggctctat
     3601 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3661 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3721 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3781 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3841 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3901 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3961 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4021 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     4081 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4141 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4201 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4261 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4321 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4381 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4441 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4501 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4561 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4621 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4681 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4741 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4801 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4861 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4921 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4981 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     5041 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5101 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5161 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5221 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5281 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5341 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5401 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5461 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5521 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5581 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5641 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5701 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5761 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5821 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5881 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5941 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6001 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6061 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6481 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6541 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6601 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6661 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6721 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6781 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6841 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6901 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6961 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7021 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7081 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7141 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7201 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7261 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7321 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7381 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7441 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7501 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7561 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7621 cgacacggaa atgttgaata ctcat
//
