LOCUS       sequence-221647-        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, 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 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.2012.09"; journalName: "Dev Cell"; date: 
            "2012-10-16- 16"; volume: "23"; issue: "4"; pages: "745-55"
COMMENT     Sequence Label: pLAP-BUBR1
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        80..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(632..634,635..637,638..1348)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(677..698)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(938..957)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1285..1306
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1394..1414
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1427..1471
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4795..4916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4832..4851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4886..4905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4923..5378)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5060..5081)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5272..5291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5405..5509
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5511..5868
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5511..5702
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5534..5554)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5719..5854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5903..6697
                     /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             5903..6697
                     /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(5957..5976)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6567..6586
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6885..6904)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6929..6976
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7305..7893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7794..7813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
      601 gtcagatccg ctagcgctac cggtcgccac catggtgagc aagggcgagg agctgttcac
      661 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      721 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      781 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca
      841 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      901 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
      961 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1021 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1081 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1141 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1201 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa
     1261 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1321 cactctcggc atggacgagc tgtacaagtc cggaggggat cggtggagct ccaccggtgg
     1381 cggccgctct agagagaatc tttattttca gggcgccgcc aaattcaaag aaaccgctgc
     1441 tgctaaattc gaacgccagc acatggacag cggaggtgga ggtactagtc tcgacgcggc
     1501 ggtgaagaag gaagggggtg ctctgagtga agccatgtcc ctggagggag atgaatggga
     1561 actgagtaaa gaaaatgtac aacctttaag gcaagggcgg atcatgtcca cgcttcaggg
     1621 agcactggca caagaatctg cctgtaacaa tactcttcag cagcagaaac gggcatttga
     1681 atatgaaatt cgattttaca ctggaaatga ccctctggat gtttgggata ggtatatcag
     1741 ctggacagag cagaactatc ctcaaggtgg gaaggagagt aatatgtcaa cgttattaga
     1801 aagagctgta gaagcactac aaggagaaaa acgatattat agtgatcctc gatttctcaa
     1861 tctctggctt aaattagggc gtttatgcaa tgagcctttg gatatgtaca gttacttgca
     1921 caaccaaggg attggtgttt cacttgctca gttctatatc tcatgggcag aagaatatga
     1981 agctagagaa aactttagga aagcagatgc gatatttcag gaagggattc aacagaaggc
     2041 tgaaccacta gaaagactac agtcccagca ccgacaattc caagctcgag tgtctcggca
     2101 aactctgttg gcacttgaga aagaagaaga ggaggaagtt tttgagtctt ctgtaccaca
     2161 acgaagcaca ctagctgaac taaagagcaa agggaaaaag acagcaagag ctccaatcat
     2221 ccgtgtagga ggtgctctca aggctccaag ccagaacaga ggactccaaa atccatttcc
     2281 tcaacagatg caaaataata gtagaattac tgtttttgat gaaaatgctg atgaggcttc
     2341 tacagcagag ttgtctaagc ctacagtcca gccatggata gcacccccca tgcccagggc
     2401 caaagagaat gagctgcaag caggcccttg gaacacaggc aggtccttgg aacacaggcc
     2461 tcgtggcaat acagcttcac tgatagctgt acccgctgtg cttcccagtt tcactccata
     2521 tgtggaagag actgcacaac agccagttat gacaccatgt aaaattgaac ctagtataaa
     2581 ccacatccta agcaccagaa agcctggaaa ggaagaagga gatcctctac aaagggttca
     2641 gagccatcag caagcatctg aggagaagaa agagaagatg atgtattgta aggagaagat
     2701 ttatgcagga gtaggggaat tctcctttga agaaattcgg gctgaagttt tccggaagaa
     2761 attaaaagag caaagggaag ccgagctatt gaccagtgca gagaagagag cagaaatgca
     2821 gaaacagatt gaagagatgg agaagaagct aaaagaaatc caaactactc agcaagaaag
     2881 aacaggtgat cagcaagaag agacgatgcc tacaaaggag acaactaaac tgcaaattgc
     2941 ttccgagtct cagaaaatac caggaatgac tctatccagt tctgtttgtc aagtaaactg
     3001 ttgtgccaga gaaacttcac ttgcggagaa catttggcag gaacaacctc attctaaagg
     3061 tcccagtgta cctttctcca tttttgatga gtttcttctt tcagaaaaga agaataaaag
     3121 tcctcctgca gatcccccac gagttttagc tcaacgaaga ccccttgcag ttctcaaaac
     3181 ctcagaaagc atcacctcaa atgaagatgt gtctccagat gtttgtgatg aatttacagg
     3241 aattgaaccc ttgagcgagg atgccattat cacaggcttc agaaatgtaa caatttgtcc
     3301 taacccagaa gacacttgtg actttgccag agcagctcgt tttgtatcca ctccttttca
     3361 tgagataatg tccttgaagg atctcccttc tgatcctgag agactgttac cggaagaaga
     3421 tctagatgta aagacctctg aggaccagca gacagcttgt ggcactatct acagtcagac
     3481 tctcagcatc aagaagctga gcccaattat tgaagacagt cgtgaagcca cacactcctc
     3541 tggcttctct ggttcttctg cctcggttgc aagcacctcc tccatcaaat gtcttcaaat
     3601 tcctgagaaa ctagaactta ctaatgagac ttcagaaaac cctactcagt caccatggtg
     3661 ttcacagtat cgcagacagc tactgaagtc cctaccagag ttaagtgcct ctgcagagtt
     3721 gtgtatagaa gacagaccaa tgcctaagtt ggaaattgag aaggaaattg aattaggtaa
     3781 tgaggattac tgcattaaac gagaatacct aatatgtgaa gattacaagt tattctgggt
     3841 ggcgccaaga aactctgcag aattaacagt aataaaggta tcttctcaac ctgtcccatg
     3901 ggacttttat atcaacctca agttaaagga acgtttaaat gaagattttg atcatttttg
     3961 cagctgttat caatatcaag atggctgtat tgtttggcac caatatataa actgcttcac
     4021 ccttcaggat cttctccaac acagtgaata tattacccat gaaataacag tgttgattat
     4081 ttataacctt ttgacaatag tggagatgct acacaaagca gaaatagtcc atggtgactt
     4141 gagtccaagg tgtctgattc tcagaaacag aatccacgat ccctatgatt gtaacaagaa
     4201 caatcaagct ttgaagatag tggacttttc ctacagtgtt gaccttaggg tgcagctgga
     4261 tgtttttacc ctcagcggct ttcggactgt acagatcctg gaaggacaaa agatactagc
     4321 taactgttct tctccctacc aggtagacct gtttggtata gcagatttag cacatttact
     4381 attgttcaag gaacacctac aggtcttctg ggatgggtcc ttctggaaac ttagccaaaa
     4441 tatttctgag ctaaaagatg gtgaattgtg gaataaattc tttgtgcgga ttctgaatgc
     4501 caatgatgag gccacagtgt ctgttcttgg ggagcttgca gcagaaatga atggggtttt
     4561 tgacactaca ttccaaagtc acctgaacaa agccttatgg aaggtaggga agttaactag
     4621 tcctggggct ttgctctttc agtgagcggc cgctctagaa ttcggggatc caccggatct
     4681 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     4741 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     4801 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4861 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     4921 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     4981 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5041 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5101 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5161 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5221 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5281 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5341 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     5401 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5461 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     5521 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     5581 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     5641 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     5701 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     5761 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     5821 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     5881 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     5941 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     6001 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     6061 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     6121 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     6181 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     6241 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     6301 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     6361 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     6421 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     6481 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     6541 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     6601 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     6661 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     6721 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     6781 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     6841 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     6901 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     6961 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     7021 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     7081 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     7141 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7201 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7261 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7321 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7381 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7441 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7501 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7561 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7621 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7681 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7741 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7801 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7861 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7921 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7981 gtggataa
//
