LOCUS       sequence-215344-        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        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1375..1395
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1408..1452
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4776..4897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4813..4832)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4867..4886
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4904..5359)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5041..5062)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5253..5272
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5386..5490
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5492..5849
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5492..5683
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5515..5535)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5700..5835
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5884..6678
                     /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             5884..6678
                     /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(5938..5957)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6548..6567
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6866..6885)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6910..6957
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7286..7874
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7775..7794
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggagggga tcggtggagc tccaccggtg gcggccgctc tagagagaat
     1381 ctttattttc agggcgccgc caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag
     1441 cacatggaca gcggaggtgg aggtactagt ctcgacgcgg cggtgaagaa ggaagggggt
     1501 gctctgagtg aagccatgtc cctggaggga gatgaatggg aactgagtaa agaaaatgta
     1561 caacctttaa ggcaagggcg gatcatgtcc acgcttcagg gagcactggc acaagaatct
     1621 gcctgtaaca atactcttca gcagcagaaa cgggcatttg aatatgaaat tcgattttac
     1681 actggaaatg accctctgga tgtttgggat aggtatatca gctggacaga gcagaactat
     1741 cctcaaggtg ggaaggagag taatatgtca acgttattag aaagagctgt agaagcacta
     1801 caaggagaaa aacgatatta tagtgatcct cgatttctca atctctggct taaattaggg
     1861 cgtttatgca atgagccttt ggatatgtac agttacttgc acaaccaagg gattggtgtt
     1921 tcacttgctc agttctatat ctcatgggca gaagaatatg aagctagaga aaactttagg
     1981 aaagcagatg cgatatttca ggaagggatt caacagaagg ctgaaccact agaaagacta
     2041 cagtcccagc accgacaatt ccaagctcga gtgtctcggc aaactctgtt ggcacttgag
     2101 aaagaagaag aggaggaagt ttttgagtct tctgtaccac aacgaagcac actagctgaa
     2161 ctaaagagca aagggaaaaa gacagcaaga gctccaatca tccgtgtagg aggtgctctc
     2221 aaggctccaa gccagaacag aggactccaa aatccatttc ctcaacagat gcaaaataat
     2281 agtagaatta ctgtttttga tgaaaatgct gatgaggctt ctacagcaga gttgtctaag
     2341 cctacagtcc agccatggat agcacccccc atgcccaggg ccaaagagaa tgagctgcaa
     2401 gcaggccctt ggaacacagg caggtccttg gaacacaggc ctcgtggcaa tacagcttca
     2461 ctgatagctg tacccgctgt gcttcccagt ttcactccat atgtggaaga gactgcacaa
     2521 cagccagtta tgacaccatg taaaattgaa cctagtataa accacatcct aagcaccaga
     2581 aagcctggaa aggaagaagg agatcctcta caaagggttc agagccatca gcaagcatct
     2641 gaggagaaga aagagaagat gatgtattgt aaggagaaga tttatgcagg agtaggggaa
     2701 ttctcctttg aagaaattcg ggctgaagtt ttccggaaga aattaaaaga gcaaagggaa
     2761 gccgagctat tgaccagtgc agagaagaga gcagaaatgc agaaacagat tgaagagatg
     2821 gagaagaagc taaaagaaat ccaaactact cagcaagaaa gaacaggtga tcagcaagaa
     2881 gagacgatgc ctacaaagga gacaactaaa ctgcaaattg cttccgagtc tcagaaaata
     2941 ccaggaatga ctctatccag ttctgtttgt caagtaaact gttgtgccag agaaacttca
     3001 cttgcggaga acatttggca ggaacaacct cattctaaag gtcccagtgt acctttctcc
     3061 atttttgatg agtttcttct ttcagaaaag aagaataaaa gtcctcctgc agatccccca
     3121 cgagttttag ctcaacgaag accccttgca gttctcaaaa cctcagaaag catcacctca
     3181 aatgaagatg tgtctccaga tgtttgtgat gaatttacag gaattgaacc cttgagcgag
     3241 gatgccatta tcacaggctt cagaaatgta acaatttgtc ctaacccaga agacacttgt
     3301 gactttgcca gagcagctcg ttttgtatcc actccttttc atgagataat gtccttgaag
     3361 gatctccctt ctgatcctga gagactgtta ccggaagaag atctagatgt aaagacctct
     3421 gaggaccagc agacagcttg tggcactatc tacagtcaga ctctcagcat caagaagctg
     3481 agcccaatta ttgaagacag tcgtgaagcc acacactcct ctggcttctc tggttcttct
     3541 gcctcggttg caagcacctc ctccatcaaa tgtcttcaaa ttcctgagaa actagaactt
     3601 actaatgaga cttcagaaaa ccctactcag tcaccatggt gttcacagta tcgcagacag
     3661 ctactgaagt ccctaccaga gttaagtgcc tctgcagagt tgtgtataga agacagacca
     3721 atgcctaagt tggaaattga gaaggaaatt gaattaggta atgaggatta ctgcattaaa
     3781 cgagaatacc taatatgtga agattacaag ttattctggg tggcgccaag aaactctgca
     3841 gaattaacag taataaaggt atcttctcaa cctgtcccat gggactttta tatcaacctc
     3901 aagttaaagg aacgtttaaa tgaagatttt gatcattttt gcagctgtta tcaatatcaa
     3961 gatggctgta ttgtttggca ccaatatata aactgcttca cccttcagga tcttctccaa
     4021 cacagtgaat atattaccca tgaaataaca gtgttgatta tttataacct tttgacaata
     4081 gtggagatgc tacacaaagc agaaatagtc catggtgact tgagtccaag gtgtctgatt
     4141 ctcagaaaca gaatccacga tccctatgat tgtaacaaga acaatcaagc tttgaagata
     4201 gtggactttt cctacagtgt tgaccttagg gtgcagctgg atgtttttac cctcagcggc
     4261 tttcggactg tacagatcct ggaaggacaa aagatactag ctaactgttc ttctccctac
     4321 caggtagacc tgtttggtat agcagattta gcacatttac tattgttcaa ggaacaccta
     4381 caggtcttct gggatgggtc cttctggaaa cttagccaaa atatttctga gctaaaagat
     4441 ggtgaattgt ggaataaatt ctttgtgcgg attctgaatg ccaatgatga ggccacagtg
     4501 tctgttcttg gggagcttgc agcagaaatg aatggggttt ttgacactac attccaaagt
     4561 cacctgaaca aagccttatg gaaggtaggg aagttaacta gtcctggggc tttgctcttt
     4621 cagtgagcgg ccgctctaga attcggggat ccaccggatc tagataactg atcataatca
     4681 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     4741 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     4801 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4861 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat
     4921 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4981 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5041 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5101 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5161 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5221 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5281 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     5341 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5401 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5461 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     5521 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5581 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     5641 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     5701 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     5761 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     5821 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     5881 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     5941 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     6001 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     6061 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     6121 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     6181 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     6241 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     6301 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     6361 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     6421 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     6481 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     6541 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     6601 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     6661 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     6721 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     6781 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     6841 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     6901 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     6961 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     7021 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     7081 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     7141 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     7201 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     7261 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     7321 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     7381 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     7441 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7501 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7561 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7621 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7681 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7741 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7801 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7861 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7921 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7981 ccatgcat
//
