LOCUS       sequence-221687-        7988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transfection in mammalian cells, combination of fluorescent protein 
            fusion and tandem affinity purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7988)
  AUTHORS   Suijkerbuijk SJ, van Dam TJ, Karagoz GE, von Castelmur E, Hubner NC,
            Duarte AM, Vleugel M, Perrakis A, Rudiger SG, Snel B, Kops GJ
  TITLE     The vertebrate mitotic checkpoint protein BUBR1 is an unusual 
            pseudokinase.
  JOURNAL   Dev Cell. 2012 Jun 12;22(6):1321-9. doi: 
            10.1016/j.devcel.2012.03.009.
  PUBMED    22698286
REFERENCE   2  (bases 1 to 7988)
  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: 
            "10.1016/j.devcel.2012.03"; journalName: "Dev Cell"; date: 
            "2012-06-12- 12"; volume: "22"; issue: "6"; pages: "1321-9"
COMMENT     Sequence Label: pLAP-BUBR1-D882N
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        106..409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        410..613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     564..584
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(658..660,661..663,664..1374)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(703..724)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(964..983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1311..1332
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1420..1440
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1453..1497
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4821..4942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4858..4877)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4912..4931
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4949..5404)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5086..5107)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5298..5317
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5431..5535
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5537..5894
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5537..5728
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5560..5580)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5745..5880
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5929..6723
                     /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             5929..6723
                     /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(5983..6002)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6593..6612
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6911..6930)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6955..7002
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7331..7919
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7820..7839
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
       61 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      421 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      481 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      541 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      601 tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg
      661 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      721 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      781 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      841 gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
      901 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      961 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1021 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1081 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1141 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1201 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1261 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1321 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
     1381 ggggatcggt ggagctccac cggtggcggc cgctctagag agaatcttta ttttcagggc
     1441 gccgccaaat tcaaagaaac cgctgctgct aaattcgaac gccagcacat ggacagcgga
     1501 ggtggaggta ctagtctcga cgcggcggtg aagaaggaag ggggtgctct gagtgaagcc
     1561 atgtccctgg agggagatga atgggaactg agtaaagaaa atgtacaacc tttaaggcaa
     1621 gggcggatca tgtccacgct tcagggagca ctggcacaag aatctgcctg taacaatact
     1681 cttcagcagc agaaacgggc atttgaatat gaaattcgat tttacactgg aaatgaccct
     1741 ctggatgttt gggataggta tatcagctgg acagagcaga actatcctca aggtgggaag
     1801 gagagtaata tgtcaacgtt attagaaaga gctgtagaag cactacaagg agaaaaacga
     1861 tattatagtg atcctcgatt tctcaatctc tggcttaaat tagggcgttt atgcaatgag
     1921 cctttggata tgtacagtta cttgcacaac caagggattg gtgtttcact tgctcagttc
     1981 tatatctcat gggcagaaga atatgaagct agagaaaact ttaggaaagc agatgcgata
     2041 tttcaggaag ggattcaaca gaaggctgaa ccactagaaa gactacagtc ccagcaccga
     2101 caattccaag ctcgagtgtc tcggcaaact ctgttggcac ttgagaaaga agaagaggag
     2161 gaagtttttg agtcttctgt accacaacga agcacactag ctgaactaaa gagcaaaggg
     2221 aaaaagacag caagagctcc aatcatccgt gtaggaggtg ctctcaaggc tccaagccag
     2281 aacagaggac tccaaaatcc atttcctcaa cagatgcaaa ataatagtag aattactgtt
     2341 tttgatgaaa atgctgatga ggcttctaca gcagagttgt ctaagcctac agtccagcca
     2401 tggatagcac cccccatgcc cagggccaaa gagaatgagc tgcaagcagg cccttggaac
     2461 acaggcaggt ccttggaaca caggcctcgt ggcaatacag cttcactgat agctgtaccc
     2521 gctgtgcttc ccagtttcac tccatatgtg gaagagactg cacaacagcc agttatgaca
     2581 ccatgtaaaa ttgaacctag tataaaccac atcctaagca ccagaaagcc tggaaaggaa
     2641 gaaggagatc ctctacaaag ggttcagagc catcagcaag catctgagga gaagaaagag
     2701 aagatgatgt attgtaagga gaagatttat gcaggagtag gggaattctc ctttgaagaa
     2761 attcgggctg aagttttccg gaagaaatta aaagagcaaa gggaagccga gctattgacc
     2821 agtgcagaga agagagcaga aatgcagaaa cagattgaag agatggagaa gaagctaaaa
     2881 gaaatccaaa ctactcagca agaaagaaca ggtgatcagc aagaagagac gatgcctaca
     2941 aaggagacaa ctaaactgca aattgcttcc gagtctcaga aaataccagg aatgactcta
     3001 tccagttctg tttgtcaagt aaactgttgt gccagagaaa cttcacttgc ggagaacatt
     3061 tggcaggaac aacctcattc taaaggtccc agtgtacctt tctccatttt tgatgagttt
     3121 cttctttcag aaaagaagaa taaaagtcct cctgcagatc ccccacgagt tttagctcaa
     3181 cgaagacccc ttgcagttct caaaacctca gaaagcatca cctcaaatga agatgtgtct
     3241 ccagatgttt gtgatgaatt tacaggaatt gaacccttga gcgaggatgc cattatcaca
     3301 ggcttcagaa atgtaacaat ttgtcctaac ccagaagaca cttgtgactt tgccagagca
     3361 gctcgttttg tatccactcc ttttcatgag ataatgtcct tgaaggatct cccttctgat
     3421 cctgagagac tgttaccgga agaagatcta gatgtaaaga cctctgagga ccagcagaca
     3481 gcttgtggca ctatctacag tcagactctc agcatcaaga agctgagccc aattattgaa
     3541 gacagtcgtg aagccacaca ctcctctggc ttctctggtt cttctgcctc ggttgcaagc
     3601 acctcctcca tcaaatgtct tcaaattcct gagaaactag aacttactaa tgagacttca
     3661 gaaaacccta ctcagtcacc atggtgttca cagtatcgca gacagctact gaagtcccta
     3721 ccagagttaa gtgcctctgc agagttgtgt atagaagaca gaccaatgcc taagttggaa
     3781 attgagaagg aaattgaatt aggtaatgag gattactgca ttaaacgaga atacctaata
     3841 tgtgaagatt acaagttatt ctgggtggcg ccaagaaact ctgcagaatt aacagtaata
     3901 aaggtatctt ctcaacctgt cccatgggac ttttatatca acctcaagtt aaaggaacgt
     3961 ttaaatgaag attttgatca tttttgcagc tgttatcaat atcaagatgg ctgtattgtt
     4021 tggcaccaat atataaactg cttcaccctt caggatcttc tccaacacag tgaatatatt
     4081 acccatgaaa taacagtgtt gattatttat aaccttttga caatagtgga gatgctacac
     4141 aaagcagaaa tagtccatgg taacttgagt ccaaggtgtc tgattctcag aaacagaatc
     4201 cacgatccct atgattgtaa caagaacaat caagctttga agatagtgga cttttcctac
     4261 agtgttgacc ttagggtgca gctggatgtt tttaccctca gcggctttcg gactgtacag
     4321 atcctggaag gacaaaagat actagctaac tgttcttctc cctaccaggt agacctgttt
     4381 ggtatagcag atttagcaca tttactattg ttcaaggaac acctacaggt cttctgggat
     4441 gggtccttct ggaaacttag ccaaaatatt tctgagctaa aagatggtga attgtggaat
     4501 aaattctttg tgcggattct gaatgccaat gatgaggcca cagtgtctgt tcttggggag
     4561 cttgcagcag aaatgaatgg ggtttttgac actacattcc aaagtcacct gaacaaagcc
     4621 ttatggaagg tagggaagtt aactagtcct ggggctttgc tctttcagtg agcggccgct
     4681 ctagaattcg gggatccacc ggatctagat aactgatcat aatcagccat accacatttg
     4741 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     4801 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     4861 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4921 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     4981 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     5041 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5101 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     5161 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     5221 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     5281 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     5341 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5401 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5461 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5521 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     5581 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5641 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5701 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     5761 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     5821 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     5881 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     5941 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     6001 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     6061 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     6121 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     6181 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     6241 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     6301 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     6361 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     6421 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     6481 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     6541 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     6601 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     6661 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     6721 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     6781 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     6841 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     6901 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     6961 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     7021 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     7081 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     7141 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     7201 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     7261 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     7321 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     7381 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     7441 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     7501 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     7561 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     7621 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     7681 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     7741 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7801 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7861 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7921 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7981 tctttcct
//
