LOCUS       sequence-123881-        7704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes kinase dead PLK4 isoform 1 carrying K41M and 
            S285A/T289A mutations with an N-terminal EGFP tag and C-terminal 
            3xFLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7704)
  AUTHORS   Sillibourne JE, Tack F, Vloemans N, Boeckx A, Thambirajah S, Bonnet 
            P, Ramaekers FC, Bornens M, Grand-Perret T
  TITLE     Autophosphorylation of polo-like kinase 4 and its role in centriole 
            duplication.
  JOURNAL   Mol Biol Cell. 2010 Feb 15;21(4):547-61. doi: 
            10.1091/mbc.E09-06-0505. Epub 2009 Dec 23.
  PUBMED    20032307
REFERENCE   2  (bases 1 to 7704)
  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: 
            "10.1091/mbc.E09-06-0505"; journalName: "Mol Biol Cell"; date: 
            "2010-02-15- 15"; volume: "21"; issue: "4"; pages: "547-61"
COMMENT     Sequence Label: pEGFP-C3-PLK4-K41M-S285A-T289A-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2478..2487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3685..3750
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    3880..4001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3917..3936)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3971..3990
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4008..4463)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4145..4166)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4357..4376
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4490..4594
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4596..4953
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4596..4787
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4619..4639)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4804..4939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4988..5782
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4988..5782
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5042..5061)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5652..5671
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5970..5989)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6014..6061
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6390..6978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6879..6898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7153..7456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7457..7660
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7611..7631
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7699..7704,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtac
      721 tcagatctcg agctcaagct tggtaccatg gcgacctgca tcggggagaa gatcgaggat
      781 tttaaagttg gaaatctgct tggtaaagga tcatttgctg gtgtctacag agctgagtcc
      841 attcacactg gtttggaagt tgcaatcatg atgatagata agaaagccat gtacaaagca
      901 ggaatggtac agagagtcca aaatgaggtg aaaatacatt gccaattgaa acatccttct
      961 atcttggagc tttataacta ttttgaagat agcaattatg tgtatctggt attagaaatg
     1021 tgccataatg gagaaatgaa caggtatcta aagaatagag tgaaaccctt ctcagaaaat
     1081 gaagctcgac acttcatgca ccagatcatc acagggatgt tgtatcttca ttctcatggt
     1141 atactacacc gggacctcac actttctaac ctcctactga ctcgtaatat gaacatcaag
     1201 attgctgatt ttgggctggc aactcaactg aaaatgccac atgaaaagca ctatacatta
     1261 tgtggaactc ctaactacat ttcaccagaa attgccactc gaagtgcaca tggccttgaa
     1321 tctgatgttt ggtccctggg ctgtatgttt tatacattac ttatcgggag accacccttc
     1381 gacactgaca cagtcaagaa cacattaaat aaagtagtat tggcagatta tgaaatgcca
     1441 tcttttttgt caatagaggc caaggacctt attcaccagt tacttcgtag aaatccagca
     1501 gatcgtttaa gtctgtcttc agtattggac catcctttta tgtcccgaaa ttcttcaaca
     1561 aaaagtaaag atttaggaac tgtggaagac tcaattgatg ctgggcatgc cgcaatttct
     1621 actgcaatta cagcttcttc cagtaccagt ataagtggta gtttatttga caaaagaaga
     1681 cttttgattg gtcagccact cccaaataaa atgactgtat ttccaaagaa taaaagttca
     1741 actgattttt cttcttcagg agatggaaac agtttttata ctcagtgggg aaatcaagaa
     1801 accagtaata gtggaagggg aagagtaatt caagatgcag aagaaaggcc acattctcga
     1861 taccttcgta gagcttattc ctctgataga tctggcactt ctaatagtca gtctcaagca
     1921 aaaacatata caatggaacg atgtcactca gcagaaatgc tttcagtgtc caaaagatca
     1981 ggaggaggtg aaaatgaaga gaggtactca cccacagaca acaatgccaa catttttaac
     2041 ttctttaaag aaaagacatc cagtagttct ggatcttttg aaagacctga taacaatcaa
     2101 gcactctcca atcatctttg tccaggaaaa actccttttc catttgcaga cccgacacct
     2161 cagactgaaa ccgtacaaca gtggtttggg aatctgcaaa taaatgctca tttaagaaaa
     2221 actactgaat atgacagcat cagcccaaac cgggacttcc agggccatcc agatttgcag
     2281 aaggacacat caaaaaatgc ctggactgat acaaaagtca aaaagaactc tgatgcttct
     2341 gataatgcac attctgtaaa acagcaaaat accatgaaat atatgactgc acttcacagt
     2401 aaacctgaga taatccaaca agaatgtgtt tttggctcag atcctctttc tgaacagagc
     2461 aagactaggg gtatggagcc accatggggt tatcagaatc gtacattaag aagcattaca
     2521 tctccgttgg ttgctcacag gttaaaacca atcagacaga aaaccaaaaa ggctgtggtg
     2581 agcatacttg attcagagga ggtgtgtgtg gagcttgtaa aggagtatgc atctcaagaa
     2641 tatgtgaaag aagttcttca gatatctagt gatggaaata cgatcactat ttattatcca
     2701 aatggtggta gaggttttcc tcttgctgat agaccaccct cacctactga caacatcagt
     2761 aggtacagct ttgacaattt accagaaaaa tactggcgaa aatatcaata tgcttccagg
     2821 tttgtacagc ttgtaagatc taaatctccc aaaatcactt attttacaag atatgctaaa
     2881 tgcattttga tggagaattc tcctggtgct gattttgagg tttggtttta tgatggggta
     2941 aaaatacaca aaacagaaga tttcattcag gtgattgaaa agacagggaa gtcttacact
     3001 ttaaaaagtg aaagtgaagt taatagcttg aaagaggaga taaaaatgta tatggaccat
     3061 gctaatgagg gtcatcgtat ttgtttagca ctggaatcca taatttcaga agaggaaagg
     3121 aaaactagga gtgctccctt tttcccaata atcataggaa gaaaacctgg tagtactagt
     3181 tcacctaagg ccttatcacc tcctccttct gtggactcaa attacccaac gagagataga
     3241 gcatctttca acagaatggt catgcatagt gctgcttctc caacacaggc accaatcctt
     3301 aatccctcta tggttacaaa tgaaggactt ggtcttacaa ctacagcttc tggaacagac
     3361 atctcttcta atagtctaaa agattgtctt cctaaatcag cacaactttt gaaatctgtt
     3421 tttgtgaaaa atgttggttg ggctacacag ttaactagtg gagctgtgtg ggttcagttt
     3481 aatgatgggt cccagttggt tgtgcaggca ggagtgtctt ctatcagtta tacctcacca
     3541 aatggtcaaa caactaggta tggagaaaat gaaaaattac cagactacat caaacagaaa
     3601 ttacagtgtc tgtcttccat ccttttgatg ttttctaatc cgactcctaa ttttcataag
     3661 cttcgaattc tgcagtcgac gatggactac aaagaccatg acggtgatta taaagatcat
     3721 gatatcgatt acaaggatga cgatgacaaa ggatccaccg gatctagata actgatcata
     3781 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3841 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     3901 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3961 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     4021 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4081 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4141 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4201 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4261 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4321 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4381 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4441 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4501 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4561 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     4621 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4681 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4741 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4801 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4861 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4921 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     4981 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     5041 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     5101 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     5161 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     5221 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     5281 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     5341 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     5401 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     5461 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     5521 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5581 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5641 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5701 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5761 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     5821 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5881 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5941 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     6001 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     6061 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     6121 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     6181 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     6241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     6541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7021 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7081 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     7141 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     7201 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     7261 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     7321 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     7381 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     7441 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     7501 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     7561 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     7621 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
     7681 ccgctagcgc taccggtcgc cacc
//
