LOCUS       sequence-221685-        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-K795A
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(615..617,618..620,621..1331)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(660..681)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(921..940)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1268..1289
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1377..1397
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1410..1454
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4778..4899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4815..4834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4869..4888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4906..5361)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5043..5064)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5255..5274
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5388..5492
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5494..5851
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5494..5685
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5517..5537)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5702..5837
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5886..6680
                     /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             5886..6680
                     /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(5940..5959)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6550..6569
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6868..6887)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6912..6959
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7288..7876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7777..7796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      661 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      721 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      781 tgccctggcc caccctcgtg accaccttcg gctacggcct gcagtgcttc gcccgctacc
      841 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      901 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      961 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1021 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1081 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1141 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1201 tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc
     1261 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1321 agctgtacaa gtccggaggg gatcggtgga gctccaccgg tggcggccgc tctagagaga
     1381 atctttattt tcagggcgcc gccaaattca aagaaaccgc tgctgctaaa ttcgaacgcc
     1441 agcacatgga cagcggaggt ggaggtacta gtctcgacgc ggcggtgaag aaggaagggg
     1501 gtgctctgag tgaagccatg tccctggagg gagatgaatg ggaactgagt aaagaaaatg
     1561 tacaaccttt aaggcaaggg cggatcatgt ccacgcttca gggagcactg gcacaagaat
     1621 ctgcctgtaa caatactctt cagcagcaga aacgggcatt tgaatatgaa attcgatttt
     1681 acactggaaa tgaccctctg gatgtttggg ataggtatat cagctggaca gagcagaact
     1741 atcctcaagg tgggaaggag agtaatatgt caacgttatt agaaagagct gtagaagcac
     1801 tacaaggaga aaaacgatat tatagtgatc ctcgatttct caatctctgg cttaaattag
     1861 ggcgtttatg caatgagcct ttggatatgt acagttactt gcacaaccaa gggattggtg
     1921 tttcacttgc tcagttctat atctcatggg cagaagaata tgaagctaga gaaaacttta
     1981 ggaaagcaga tgcgatattt caggaaggga ttcaacagaa ggctgaacca ctagaaagac
     2041 tacagtccca gcaccgacaa ttccaagctc gagtgtctcg gcaaactctg ttggcacttg
     2101 agaaagaaga agaggaggaa gtttttgagt cttctgtacc acaacgaagc acactagctg
     2161 aactaaagag caaagggaaa aagacagcaa gagctccaat catccgtgta ggaggtgctc
     2221 tcaaggctcc aagccagaac agaggactcc aaaatccatt tcctcaacag atgcaaaata
     2281 atagtagaat tactgttttt gatgaaaatg ctgatgaggc ttctacagca gagttgtcta
     2341 agcctacagt ccagccatgg atagcacccc ccatgcccag ggccaaagag aatgagctgc
     2401 aagcaggccc ttggaacaca ggcaggtcct tggaacacag gcctcgtggc aatacagctt
     2461 cactgatagc tgtacccgct gtgcttccca gtttcactcc atatgtggaa gagactgcac
     2521 aacagccagt tatgacacca tgtaaaattg aacctagtat aaaccacatc ctaagcacca
     2581 gaaagcctgg aaaggaagaa ggagatcctc tacaaagggt tcagagccat cagcaagcat
     2641 ctgaggagaa gaaagagaag atgatgtatt gtaaggagaa gatttatgca ggagtagggg
     2701 aattctcctt tgaagaaatt cgggctgaag ttttccggaa gaaattaaaa gagcaaaggg
     2761 aagccgagct attgaccagt gcagagaaga gagcagaaat gcagaaacag attgaagaga
     2821 tggagaagaa gctaaaagaa atccaaacta ctcagcaaga aagaacaggt gatcagcaag
     2881 aagagacgat gcctacaaag gagacaacta aactgcaaat tgcttccgag tctcagaaaa
     2941 taccaggaat gactctatcc agttctgttt gtcaagtaaa ctgttgtgcc agagaaactt
     3001 cacttgcgga gaacatttgg caggaacaac ctcattctaa aggtcccagt gtacctttct
     3061 ccatttttga tgagtttctt ctttcagaaa agaagaataa aagtcctcct gcagatcccc
     3121 cacgagtttt agctcaacga agaccccttg cagttctcaa aacctcagaa agcatcacct
     3181 caaatgaaga tgtgtctcca gatgtttgtg atgaatttac aggaattgaa cccttgagcg
     3241 aggatgccat tatcacaggc ttcagaaatg taacaatttg tcctaaccca gaagacactt
     3301 gtgactttgc cagagcagct cgttttgtat ccactccttt tcatgagata atgtccttga
     3361 aggatctccc ttctgatcct gagagactgt taccggaaga agatctagat gtaaagacct
     3421 ctgaggacca gcagacagct tgtggcacta tctacagtca gactctcagc atcaagaagc
     3481 tgagcccaat tattgaagac agtcgtgaag ccacacactc ctctggcttc tctggttctt
     3541 ctgcctcggt tgcaagcacc tcctccatca aatgtcttca aattcctgag aaactagaac
     3601 ttactaatga gacttcagaa aaccctactc agtcaccatg gtgttcacag tatcgcagac
     3661 agctactgaa gtccctacca gagttaagtg cctctgcaga gttgtgtata gaagacagac
     3721 caatgcctaa gttggaaatt gagaaggaaa ttgaattagg taatgaggat tactgcatta
     3781 aacgagaata cctaatatgt gaagattaca agttattctg ggtggcgcca agaaactctg
     3841 cagaattaac agtaatagcg gtatcttctc aacctgtccc atgggacttt tatatcaacc
     3901 tcaagttaaa ggaacgttta aatgaagatt ttgatcattt ttgcagctgt tatcaatatc
     3961 aagatggctg tattgtttgg caccaatata taaactgctt cacccttcag gatcttctcc
     4021 aacacagtga atatattacc catgaaataa cagtgttgat tatttataac cttttgacaa
     4081 tagtggagat gctacacaaa gcagaaatag tccatggtga cttgagtcca aggtgtctga
     4141 ttctcagaaa cagaatccac gatccctatg attgtaacaa gaacaatcaa gctttgaaga
     4201 tagtggactt ttcctacagt gttgacctta gggtgcagct ggatgttttt accctcagcg
     4261 gctttcggac tgtacagatc ctggaaggac aaaagatact agctaactgt tcttctccct
     4321 accaggtaga cctgtttggt atagcagatt tagcacattt actattgttc aaggaacacc
     4381 tacaggtctt ctgggatggg tccttctgga aacttagcca aaatatttct gagctaaaag
     4441 atggtgaatt gtggaataaa ttctttgtgc ggattctgaa tgccaatgat gaggccacag
     4501 tgtctgttct tggggagctt gcagcagaaa tgaatggggt ttttgacact acattccaaa
     4561 gtcacctgaa caaagcctta tggaaggtag ggaagttaac tagtcctggg gctttgctct
     4621 ttcagtgagc ggccgctcta gaattcgggg atccaccgga tctagataac tgatcataat
     4681 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     4741 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     4801 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4861 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     4921 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4981 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5041 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5101 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5161 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5221 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5281 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5341 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5461 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     5521 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5581 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     5641 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5701 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5761 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5821 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     5881 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     5941 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     6001 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     6061 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     6121 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     6181 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     6241 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     6301 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     6361 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     6421 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     6481 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     6541 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     6601 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     6661 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     6721 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     6781 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     6841 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     6901 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     6961 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     7021 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     7081 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     7141 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7201 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7261 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7321 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7381 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7441 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7501 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7561 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7621 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7681 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7741 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7801 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7861 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7921 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7981 cgccatgc
//
