LOCUS       sequence-221668-        7934 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 7934)
  AUTHORS   Suijkerbuijk SJ, Vleugel M, Teixeira A, Kops GJ
  TITLE     Integration of kinase and phosphatase activities by BUBR1 ensures 
            formation of stable kinetochore-microtubule attachments.
  JOURNAL   Dev Cell. 2012 Oct 16;23(4):745-55. doi: 
            10.1016/j.devcel.2012.09.005.
  PUBMED    23079597
REFERENCE   2  (bases 1 to 7934)
  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.2012.09"; journalName: "Dev Cell"; date: 
            "2012-10-16- 16"; volume: "23"; issue: "4"; pages: "745-55"
COMMENT     Sequence Label: pLAP-BUBR1-dKARD
FEATURES             Location/Qualifiers
     source          1..7934
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        79..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..586
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     537..557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      625..634
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(631..633,634..636,637..1347)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(676..697)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(937..956)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1284..1305
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1393..1413
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1426..1470
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4740..4861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4777..4796)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4831..4850
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4868..5323)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5005..5026)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5217..5236
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5350..5454
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5456..5813
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5456..5647
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5479..5499)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5664..5799
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5848..6642
                     /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             5848..6642
                     /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(5902..5921)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6512..6531
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6830..6849)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6874..6921
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7250..7838
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7739..7758
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      421 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      481 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      541 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
      601 tcagatccgc tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
      661 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      721 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      781 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgcag
      841 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      901 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      961 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1021 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1081 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1141 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1201 gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa
     1261 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1321 actctcggca tggacgagct gtacaagtcc ggaggggatc ggtggagctc caccggtggc
     1381 ggccgctcta gagagaatct ttattttcag ggcgccgcca aattcaaaga aaccgctgct
     1441 gctaaattcg aacgccagca catggacagc ggaggtggag gtactagtct cgacgcggcg
     1501 gtgaagaagg aagggggtgc tctgagtgaa gccatgtccc tggagggaga tgaatgggaa
     1561 ctgagtaaag aaaatgtaca acctttaagg caagggcgga tcatgtccac gcttcaggga
     1621 gcactggcac aagaatctgc ctgtaacaat actcttcagc agcagaaacg ggcatttgaa
     1681 tatgaaattc gattttacac tggaaatgac cctctggatg tttgggatag gtatatcagc
     1741 tggacagagc agaactatcc tcaaggtggg aaggagagta atatgtcaac gttattagaa
     1801 agagctgtag aagcactaca aggagaaaaa cgatattata gtgatcctcg atttctcaat
     1861 ctctggctta aattagggcg tttatgcaat gagcctttgg atatgtacag ttacttgcac
     1921 aaccaaggga ttggtgtttc acttgctcag ttctatatct catgggcaga agaatatgaa
     1981 gctagagaaa actttaggaa agcagatgcg atatttcagg aagggattca acagaaggct
     2041 gaaccactag aaagactaca gtcccagcac cgacaattcc aagctcgagt gtctcggcaa
     2101 actctgttgg cacttgagaa agaagaagag gaggaagttt ttgagtcttc tgtaccacaa
     2161 cgaagcacac tagctgaact aaagagcaaa gggaaaaaga cagcaagagc tccaatcatc
     2221 cgtgtaggag gtgctctcaa ggctccaagc cagaacagag gactccaaaa tccatttcct
     2281 caacagatgc aaaataatag tagaattact gtttttgatg aaaatgctga tgaggcttct
     2341 acagcagagt tgtctaagcc tacagtccag ccatggatag caccccccat gcccagggcc
     2401 aaagagaatg agctgcaagc aggcccttgg aacacaggca ggtccttgga acacaggcct
     2461 cgtggcaata cagcttcact gatagctgta cccgctgtgc ttcccagttt cactccatat
     2521 gtggaagaga ctgcacaaca gccagttatg acaccatgta aaattgaacc tagtataaac
     2581 cacatcctaa gcaccagaaa gcctggaaag gaagaaggag atcctctaca aagggttcag
     2641 agccatcagc aagcatctga ggagaagaaa gagaagatga tgtattgtaa ggagaagatt
     2701 tatgcaggag taggggaatt ctcctttgaa gaaattcggg ctgaagtttt ccggaagaaa
     2761 ttaaaagagc aaagggaagc cgagctattg accagtgcag agaagagagc agaaatgcag
     2821 aaacagattg aagagatgga gaagaagcta aaagaaatcc aaactactca gcaagaaaga
     2881 acaggtgatc agcaagaaga gacgatgcct acaaaggaga caactaaact gcaaattgct
     2941 tccgagtctc agaaaatacc aggaatgact ctatccagtt ctgtttgtca agtaaactgt
     3001 tgtgccagag aaacttcact tgcggagaac atttggcagg aacaacctca ttctaaaggt
     3061 cccagtgtac ctttctccat ttttgatgag tttcttcttt cagaaaagaa gaataaaagt
     3121 cctcctgcag atcccccacg agttttagct caacgaagac cccttgcagt tctcaaaacc
     3181 tcagaaagca tcacctcaaa tgaagatgtg tctccagatg tttgtgatga atttacagga
     3241 attgaaccct tgagcgagga tgccattatc acaggcttca gaaatgtaac aatttgtcct
     3301 aacccagaag acacttgtga ctttgccaga gcagctcgtt ttgtatccac tccttttcat
     3361 gagataatgt ccttgaagga tctcccttct gatcctgaga gactgttacc ggaagaagat
     3421 ctagatgtaa agacctctga ggaccagcag acagcttgtg gcactatcta cagtcagact
     3481 tcctctggct tctctggttc ttctgcctcg gttgcaagca cctcctccat caaatgtctt
     3541 caaattcctg agaaactaga acttactaat gagacttcag aaaaccctac tcagtcacca
     3601 tggtgttcac agtatcgcag acagctactg aagtccctac cagagttaag tgcctctgca
     3661 gagttgtgta tagaagacag accaatgcct aagttggaaa ttgagaagga aattgaatta
     3721 ggtaatgagg attactgcat taaacgagaa tacctaatat gtgaagatta caagttattc
     3781 tgggtggcgc caagaaactc tgcagaatta acagtaataa aggtatcttc tcaacctgtc
     3841 ccatgggact tttatatcaa cctcaagtta aaggaacgtt taaatgaaga ttttgatcat
     3901 ttttgcagct gttatcaata tcaagatggc tgtattgttt ggcaccaata tataaactgc
     3961 ttcacccttc aggatcttct ccaacacagt gaatatatta cccatgaaat aacagtgttg
     4021 attatttata accttttgac aatagtggag atgctacaca aagcagaaat agtccatggt
     4081 gacttgagtc caaggtgtct gattctcaga aacagaatcc acgatcccta tgattgtaac
     4141 aagaacaatc aagctttgaa gatagtggac ttttcctaca gtgttgacct tagggtgcag
     4201 ctggatgttt ttaccctcag cggctttcgg actgtacaga tcctggaagg acaaaagata
     4261 ctagctaact gttcttctcc ctaccaggta gacctgtttg gtatagcaga tttagcacat
     4321 ttactattgt tcaaggaaca cctacaggtc ttctgggatg ggtccttctg gaaacttagc
     4381 caaaatattt ctgagctaaa agatggtgaa ttgtggaata aattctttgt gcggattctg
     4441 aatgccaatg atgaggccac agtgtctgtt cttggggagc ttgcagcaga aatgaatggg
     4501 gtttttgaca ctacattcca aagtcacctg aacaaagcct tatggaaggt agggaagtta
     4561 actagtcctg gggctttgct ctttcagtga gcggccgctc tagaattcgg ggatccaccg
     4621 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     4681 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     4741 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4801 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4861 aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4921 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4981 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5041 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5101 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5161 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5221 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5281 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     5341 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5401 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     5461 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     5521 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     5581 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5641 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     5701 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     5761 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     5821 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     5881 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     5941 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     6001 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     6061 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     6121 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     6181 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     6241 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     6301 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     6361 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     6421 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     6481 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     6541 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     6601 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     6661 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     6721 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     6781 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     6841 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     6901 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     6961 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     7021 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     7081 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7141 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7201 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7261 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7321 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7381 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7441 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7501 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7561 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7621 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7681 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7741 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7801 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7861 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7921 attctgtgga taac
//
