LOCUS       sequence-233658-        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-BUBR1-3A
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        43..346
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        347..550
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     501..521
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      589..598
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(595..597,598..600,601..1311)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(640..661)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(901..920)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1248..1269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1357..1377
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1390..1434
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4758..4879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4795..4814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4849..4868
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4886..5341)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5023..5044)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5235..5254
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5368..5472
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5474..5831
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5474..5665
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5497..5517)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5682..5817
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5866..6660
                     /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             5866..6660
                     /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(5920..5939)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6530..6549
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6848..6867)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6892..6939
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7268..7856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7757..7776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
       61 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      121 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      181 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      241 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      301 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      361 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      421 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      481 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      541 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg
      601 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      661 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      721 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      781 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
      841 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      901 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      961 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1021 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1081 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1141 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1201 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1261 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggaggg
     1321 gatcggtgga gctccaccgg tggcggccgc tctagagaga atctttattt tcagggcgcc
     1381 gccaaattca aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga cagcggaggt
     1441 ggaggtacta gtctcgacgc ggcggtgaag aaggaagggg gtgctctgag tgaagccatg
     1501 tccctggagg gagatgaatg ggaactgagt aaagaaaatg tacaaccttt aaggcaaggg
     1561 cggatcatgt ccacgcttca gggagcactg gcacaagaat ctgcctgtaa caatactctt
     1621 cagcagcaga aacgggcatt tgaatatgaa attcgatttt acactggaaa tgaccctctg
     1681 gatgtttggg ataggtatat cagctggaca gagcagaact atcctcaagg tgggaaggag
     1741 agtaatatgt caacgttatt agaaagagct gtagaagcac tacaaggaga aaaacgatat
     1801 tatagtgatc ctcgatttct caatctctgg cttaaattag ggcgtttatg caatgagcct
     1861 ttggatatgt acagttactt gcacaaccaa gggattggtg tttcacttgc tcagttctat
     1921 atctcatggg cagaagaata tgaagctaga gaaaacttta ggaaagcaga tgcgatattt
     1981 caggaaggga ttcaacagaa ggctgaacca ctagaaagac tacagtccca gcaccgacaa
     2041 ttccaagctc gagtgtctcg gcaaactctg ttggcacttg agaaagaaga agaggaggaa
     2101 gtttttgagt cttctgtacc acaacgaagc acactagctg aactaaagag caaagggaaa
     2161 aagacagcaa gagctccaat catccgtgta ggaggtgctc tcaaggctcc aagccagaac
     2221 agaggactcc aaaatccatt tcctcaacag atgcaaaata atagtagaat tactgttttt
     2281 gatgaaaatg ctgatgaggc ttctacagca gagttgtcta agcctacagt ccagccatgg
     2341 atagcacccc ccatgcccag ggccaaagag aatgagctgc aagcaggccc ttggaacaca
     2401 ggcaggtcct tggaacacag gcctcgtggc aatacagctt cactgatagc tgtacccgct
     2461 gtgcttccca gtttcactcc atatgtggaa gagactgcac aacagccagt tatgacacca
     2521 tgtaaaattg aacctagtat aaaccacatc ctaagcacca gaaagcctgg aaaggaagaa
     2581 ggagatcctc tacaaagggt tcagagccat cagcaagcat ctgaggagaa gaaagagaag
     2641 atgatgtatt gtaaggagaa gatttatgca ggagtagggg aattctcctt tgaagaaatt
     2701 cgggctgaag ttttccggaa gaaattaaaa gagcaaaggg aagccgagct attgaccagt
     2761 gcagagaaga gagcagaaat gcagaaacag attgaagaga tggagaagaa gctaaaagaa
     2821 atccaaacta ctcagcaaga aagaacaggt gatcagcaag aagagacgat gcctacaaag
     2881 gagacaacta aactgcaaat tgcttccgag tctcagaaaa taccaggaat gactctatcc
     2941 agttctgttt gtcaagtaaa ctgttgtgcc agagaaactt cacttgcgga gaacatttgg
     3001 caggaacaac ctcattctaa aggtcccagt gtacctttct ccatttttga tgagtttctt
     3061 ctttcagaaa agaagaataa aagtcctcct gcagatcccc cacgagtttt agctcaacga
     3121 agaccccttg cagttctcaa aacctcagaa agcatcacct caaatgaaga tgtgtctcca
     3181 gatgtttgtg atgaatttac aggaattgaa cccttgagcg aggatgccat tatcacaggc
     3241 ttcagaaatg taacaatttg tcctaaccca gaagacactt gtgactttgc cagagcagct
     3301 cgttttgtat ccactccttt tcatgagata atgtccttga aggatctccc ttctgatcct
     3361 gagagactgt taccggaaga agatctagat gtaaagacct ctgaggacca gcagacagct
     3421 tgtggcacta tctacagtca gactctcagc atcaagaagc tggccccaat tattgaagac
     3481 gctcgtgaag ccgcacactc ctctggcttc tctggttctt ctgcctcggt tgcaagcacc
     3541 tcctccatca aatgtcttca aattcctgag aaactagaac ttactaatga gacttcagaa
     3601 aaccctactc agtcaccatg gtgttcacag tatcgcagac agctactgaa gtccctacca
     3661 gagttaagtg cctctgcaga gttgtgtata gaagacagac caatgcctaa gttggaaatt
     3721 gagaaggaaa ttgaattagg taatgaggat tactgcatta aacgagaata cctaatatgt
     3781 gaagattaca agttattctg ggtggcgcca agaaactctg cagaattaac agtaataaag
     3841 gtatcttctc aacctgtccc atgggacttt tatatcaacc tcaagttaaa ggaacgttta
     3901 aatgaagatt ttgatcattt ttgcagctgt tatcaatatc aagatggctg tattgtttgg
     3961 caccaatata taaactgctt cacccttcag gatcttctcc aacacagtga atatattacc
     4021 catgaaataa cagtgttgat tatttataac cttttgacaa tagtggagat gctacacaaa
     4081 gcagaaatag tccatggtga cttgagtcca aggtgtctga ttctcagaaa cagaatccac
     4141 gatccctatg attgtaacaa gaacaatcaa gctttgaaga tagtggactt ttcctacagt
     4201 gttgacctta gggtgcagct ggatgttttt accctcagcg gctttcggac tgtacagatc
     4261 ctggaaggac aaaagatact agctaactgt tcttctccct accaggtaga cctgtttggt
     4321 atagcagatt tagcacattt actattgttc aaggaacacc tacaggtctt ctgggatggg
     4381 tccttctgga aacttagcca aaatatttct gagctaaaag atggtgaatt gtggaataaa
     4441 ttctttgtgc ggattctgaa tgccaatgat gaggccacag tgtctgttct tggggagctt
     4501 gcagcagaaa tgaatggggt ttttgacact acattccaaa gtcacctgaa caaagcctta
     4561 tggaaggtag ggaagttaac tagtcctggg gctttgctct ttcagtgagc ggccgctcta
     4621 gaattcgggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     4681 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     4741 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4801 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4861 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     4921 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4981 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5041 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5101 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5161 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5221 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5281 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     5341 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5401 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5461 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     5521 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     5581 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     5641 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5701 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5761 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5821 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     5881 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     5941 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     6001 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     6061 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     6121 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     6181 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     6241 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     6301 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     6361 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     6421 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     6481 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     6541 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     6601 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     6661 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     6721 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     6781 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     6841 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     6901 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     6961 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     7021 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     7081 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     7141 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7201 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7261 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7321 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7381 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7441 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7501 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7561 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7621 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7681 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7741 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7801 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7861 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7921 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa
     7981 tagtaatc
//
