LOCUS       sequence-233657-        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-BUBR1-3D
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      632..641
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(638..640,641..643,644..1354)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(683..704)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(944..963)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1291..1312
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1400..1420
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1433..1477
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4801..4922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4838..4857)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4892..4911
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4929..5384)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5066..5087)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5278..5297
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5411..5515
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5517..5874
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5517..5708
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5540..5560)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5725..5860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5909..6703
                     /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             5909..6703
                     /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(5963..5982)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6573..6592
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6891..6910)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6935..6982
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7311..7899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7800..7819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      601 tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct
      661 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      721 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      781 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg
      841 cctgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      901 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      961 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1021 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1081 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1141 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1201 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct
     1261 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1321 cgggatcact ctcggcatgg acgagctgta caagtccgga ggggatcggt ggagctccac
     1381 cggtggcggc cgctctagag agaatcttta ttttcagggc gccgccaaat tcaaagaaac
     1441 cgctgctgct aaattcgaac gccagcacat ggacagcgga ggtggaggta ctagtctcga
     1501 cgcggcggtg aagaaggaag ggggtgctct gagtgaagcc atgtccctgg agggagatga
     1561 atgggaactg agtaaagaaa atgtacaacc tttaaggcaa gggcggatca tgtccacgct
     1621 tcagggagca ctggcacaag aatctgcctg taacaatact cttcagcagc agaaacgggc
     1681 atttgaatat gaaattcgat tttacactgg aaatgaccct ctggatgttt gggataggta
     1741 tatcagctgg acagagcaga actatcctca aggtgggaag gagagtaata tgtcaacgtt
     1801 attagaaaga gctgtagaag cactacaagg agaaaaacga tattatagtg atcctcgatt
     1861 tctcaatctc tggcttaaat tagggcgttt atgcaatgag cctttggata tgtacagtta
     1921 cttgcacaac caagggattg gtgtttcact tgctcagttc tatatctcat gggcagaaga
     1981 atatgaagct agagaaaact ttaggaaagc agatgcgata tttcaggaag ggattcaaca
     2041 gaaggctgaa ccactagaaa gactacagtc ccagcaccga caattccaag ctcgagtgtc
     2101 tcggcaaact ctgttggcac ttgagaaaga agaagaggag gaagtttttg agtcttctgt
     2161 accacaacga agcacactag ctgaactaaa gagcaaaggg aaaaagacag caagagctcc
     2221 aatcatccgt gtaggaggtg ctctcaaggc tccaagccag aacagaggac tccaaaatcc
     2281 atttcctcaa cagatgcaaa ataatagtag aattactgtt tttgatgaaa atgctgatga
     2341 ggcttctaca gcagagttgt ctaagcctac agtccagcca tggatagcac cccccatgcc
     2401 cagggccaaa gagaatgagc tgcaagcagg cccttggaac acaggcaggt ccttggaaca
     2461 caggcctcgt ggcaatacag cttcactgat agctgtaccc gctgtgcttc ccagtttcac
     2521 tccatatgtg gaagagactg cacaacagcc agttatgaca ccatgtaaaa ttgaacctag
     2581 tataaaccac atcctaagca ccagaaagcc tggaaaggaa gaaggagatc ctctacaaag
     2641 ggttcagagc catcagcaag catctgagga gaagaaagag aagatgatgt attgtaagga
     2701 gaagatttat gcaggagtag gggaattctc ctttgaagaa attcgggctg aagttttccg
     2761 gaagaaatta aaagagcaaa gggaagccga gctattgacc agtgcagaga agagagcaga
     2821 aatgcagaaa cagattgaag agatggagaa gaagctaaaa gaaatccaaa ctactcagca
     2881 agaaagaaca ggtgatcagc aagaagagac gatgcctaca aaggagacaa ctaaactgca
     2941 aattgcttcc gagtctcaga aaataccagg aatgactcta tccagttctg tttgtcaagt
     3001 aaactgttgt gccagagaaa cttcacttgc ggagaacatt tggcaggaac aacctcattc
     3061 taaaggtccc agtgtacctt tctccatttt tgatgagttt cttctttcag aaaagaagaa
     3121 taaaagtcct cctgcagatc ccccacgagt tttagctcaa cgaagacccc ttgcagttct
     3181 caaaacctca gaaagcatca cctcaaatga agatgtgtct ccagatgttt gtgatgaatt
     3241 tacaggaatt gaacccttga gcgaggatgc cattatcaca ggcttcagaa atgtaacaat
     3301 ttgtcctaac ccagaagaca cttgtgactt tgccagagca gctcgttttg tatccactcc
     3361 ttttcatgag ataatgtcct tgaaggatct cccttctgat cctgagagac tgttaccgga
     3421 agaagatcta gatgtaaaga cctctgagga ccagcagaca gcttgtggca ctatctacag
     3481 tcagactctc agcatcaaga agctggaccc aattattgaa gacgatcgtg aagccgacca
     3541 ctcctctggc ttctctggtt cttctgcctc ggttgcaagc acctcctcca tcaaatgtct
     3601 tcaaattcct gagaaactag aacttactaa tgagacttca gaaaacccta ctcagtcacc
     3661 atggtgttca cagtatcgca gacagctact gaagtcccta ccagagttaa gtgcctctgc
     3721 agagttgtgt atagaagaca gaccaatgcc taagttggaa attgagaagg aaattgaatt
     3781 aggtaatgag gattactgca ttaaacgaga atacctaata tgtgaagatt acaagttatt
     3841 ctgggtggcg ccaagaaact ctgcagaatt aacagtaata aaggtatctt ctcaacctgt
     3901 cccatgggac ttttatatca acctcaagtt aaaggaacgt ttaaatgaag attttgatca
     3961 tttttgcagc tgttatcaat atcaagatgg ctgtattgtt tggcaccaat atataaactg
     4021 cttcaccctt caggatcttc tccaacacag tgaatatatt acccatgaaa taacagtgtt
     4081 gattatttat aaccttttga caatagtgga gatgctacac aaagcagaaa tagtccatgg
     4141 tgacttgagt ccaaggtgtc tgattctcag aaacagaatc cacgatccct atgattgtaa
     4201 caagaacaat caagctttga agatagtgga cttttcctac agtgttgacc ttagggtgca
     4261 gctggatgtt tttaccctca gcggctttcg gactgtacag atcctggaag gacaaaagat
     4321 actagctaac tgttcttctc cctaccaggt agacctgttt ggtatagcag atttagcaca
     4381 tttactattg ttcaaggaac acctacaggt cttctgggat gggtccttct ggaaacttag
     4441 ccaaaatatt tctgagctaa aagatggtga attgtggaat aaattctttg tgcggattct
     4501 gaatgccaat gatgaggcca cagtgtctgt tcttggggag cttgcagcag aaatgaatgg
     4561 ggtttttgac actacattcc aaagtcacct gaacaaagcc ttatggaagg tagggaagtt
     4621 aactagtcct ggggctttgc tctttcagtg agcggccgct ctagaattcg gggatccacc
     4681 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     4741 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     4801 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4861 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4921 taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4981 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5041 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5101 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5161 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5221 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5281 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5341 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     5401 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     5521 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     5581 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     5641 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     5701 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     5761 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     5821 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga
     5881 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     5941 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     6001 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     6061 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     6121 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     6181 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     6241 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     6301 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     6361 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     6421 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     6481 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     6541 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     6601 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     6661 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     6721 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     6781 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     6841 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
     6901 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     6961 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     7021 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     7081 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     7141 gccatagcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
     7201 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7921 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7981 gattctgt
//
