LOCUS       sequence-221681-        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-K795R
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..330
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        331..534
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     485..505
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      573..582
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(579..581,582..584,585..1295)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(624..645)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(885..904)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1232..1253
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1341..1361
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1374..1418
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    4742..4863
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4779..4798)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4833..4852
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4870..5325)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5007..5028)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5219..5238
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5352..5456
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5458..5815
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5458..5649
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5481..5501)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5666..5801
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5850..6644
                     /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             5850..6644
                     /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(5904..5923)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6514..6533
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6832..6851)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6876..6923
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7252..7840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7741..7760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
       61 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      121 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      181 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      241 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      301 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      361 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      421 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      481 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
      541 gtgaaccgtc agatccgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc
      601 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
      661 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
      721 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
      781 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
      841 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
      901 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
      961 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1021 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     1081 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1141 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc
     1201 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1261 ccgggatcac tctcggcatg gacgagctgt acaagtccgg aggggatcgg tggagctcca
     1321 ccggtggcgg ccgctctaga gagaatcttt attttcaggg cgccgccaaa ttcaaagaaa
     1381 ccgctgctgc taaattcgaa cgccagcaca tggacagcgg aggtggaggt actagtctcg
     1441 acgcggcggt gaagaaggaa gggggtgctc tgagtgaagc catgtccctg gagggagatg
     1501 aatgggaact gagtaaagaa aatgtacaac ctttaaggca agggcggatc atgtccacgc
     1561 ttcagggagc actggcacaa gaatctgcct gtaacaatac tcttcagcag cagaaacggg
     1621 catttgaata tgaaattcga ttttacactg gaaatgaccc tctggatgtt tgggataggt
     1681 atatcagctg gacagagcag aactatcctc aaggtgggaa ggagagtaat atgtcaacgt
     1741 tattagaaag agctgtagaa gcactacaag gagaaaaacg atattatagt gatcctcgat
     1801 ttctcaatct ctggcttaaa ttagggcgtt tatgcaatga gcctttggat atgtacagtt
     1861 acttgcacaa ccaagggatt ggtgtttcac ttgctcagtt ctatatctca tgggcagaag
     1921 aatatgaagc tagagaaaac tttaggaaag cagatgcgat atttcaggaa gggattcaac
     1981 agaaggctga accactagaa agactacagt cccagcaccg acaattccaa gctcgagtgt
     2041 ctcggcaaac tctgttggca cttgagaaag aagaagagga ggaagttttt gagtcttctg
     2101 taccacaacg aagcacacta gctgaactaa agagcaaagg gaaaaagaca gcaagagctc
     2161 caatcatccg tgtaggaggt gctctcaagg ctccaagcca gaacagagga ctccaaaatc
     2221 catttcctca acagatgcaa aataatagta gaattactgt ttttgatgaa aatgctgatg
     2281 aggcttctac agcagagttg tctaagccta cagtccagcc atggatagca ccccccatgc
     2341 ccagggccaa agagaatgag ctgcaagcag gcccttggaa cacaggcagg tccttggaac
     2401 acaggcctcg tggcaataca gcttcactga tagctgtacc cgctgtgctt cccagtttca
     2461 ctccatatgt ggaagagact gcacaacagc cagttatgac accatgtaaa attgaaccta
     2521 gtataaacca catcctaagc accagaaagc ctggaaagga agaaggagat cctctacaaa
     2581 gggttcagag ccatcagcaa gcatctgagg agaagaaaga gaagatgatg tattgtaagg
     2641 agaagattta tgcaggagta ggggaattct cctttgaaga aattcgggct gaagttttcc
     2701 ggaagaaatt aaaagagcaa agggaagccg agctattgac cagtgcagag aagagagcag
     2761 aaatgcagaa acagattgaa gagatggaga agaagctaaa agaaatccaa actactcagc
     2821 aagaaagaac aggtgatcag caagaagaga cgatgcctac aaaggagaca actaaactgc
     2881 aaattgcttc cgagtctcag aaaataccag gaatgactct atccagttct gtttgtcaag
     2941 taaactgttg tgccagagaa acttcacttg cggagaacat ttggcaggaa caacctcatt
     3001 ctaaaggtcc cagtgtacct ttctccattt ttgatgagtt tcttctttca gaaaagaaga
     3061 ataaaagtcc tcctgcagat cccccacgag ttttagctca acgaagaccc cttgcagttc
     3121 tcaaaacctc agaaagcatc acctcaaatg aagatgtgtc tccagatgtt tgtgatgaat
     3181 ttacaggaat tgaacccttg agcgaggatg ccattatcac aggcttcaga aatgtaacaa
     3241 tttgtcctaa cccagaagac acttgtgact ttgccagagc agctcgtttt gtatccactc
     3301 cttttcatga gataatgtcc ttgaaggatc tcccttctga tcctgagaga ctgttaccgg
     3361 aagaagatct agatgtaaag acctctgagg accagcagac agcttgtggc actatctaca
     3421 gtcagactct cagcatcaag aagctgagcc caattattga agacagtcgt gaagccacac
     3481 actcctctgg cttctctggt tcttctgcct cggttgcaag cacctcctcc atcaaatgtc
     3541 ttcaaattcc tgagaaacta gaacttacta atgagacttc agaaaaccct actcagtcac
     3601 catggtgttc acagtatcgc agacagctac tgaagtccct accagagtta agtgcctctg
     3661 cagagttgtg tatagaagac agaccaatgc ctaagttgga aattgagaag gaaattgaat
     3721 taggtaatga ggattactgc attaaacgag aatacctaat atgtgaagat tacaagttat
     3781 tctgggtggc gccaagaaac tctgcagaat taacagtaat aagggtatct tctcaacctg
     3841 tcccatggga cttttatatc aacctcaagt taaaggaacg tttaaatgaa gattttgatc
     3901 atttttgcag ctgttatcaa tatcaagatg gctgtattgt ttggcaccaa tatataaact
     3961 gcttcaccct tcaggatctt ctccaacaca gtgaatatat tacccatgaa ataacagtgt
     4021 tgattattta taaccttttg acaatagtgg agatgctaca caaagcagaa atagtccatg
     4081 gtgacttgag tccaaggtgt ctgattctca gaaacagaat ccacgatccc tatgattgta
     4141 acaagaacaa tcaagctttg aagatagtgg acttttccta cagtgttgac cttagggtgc
     4201 agctggatgt ttttaccctc agcggctttc ggactgtaca gatcctggaa ggacaaaaga
     4261 tactagctaa ctgttcttct ccctaccagg tagacctgtt tggtatagca gatttagcac
     4321 atttactatt gttcaaggaa cacctacagg tcttctggga tgggtccttc tggaaactta
     4381 gccaaaatat ttctgagcta aaagatggtg aattgtggaa taaattcttt gtgcggattc
     4441 tgaatgccaa tgatgaggcc acagtgtctg ttcttgggga gcttgcagca gaaatgaatg
     4501 gggtttttga cactacattc caaagtcacc tgaacaaagc cttatggaag gtagggaagt
     4561 taactagtcc tggggctttg ctctttcagt gagcggccgc tctagaattc ggggatccac
     4621 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4681 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4741 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4801 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4861 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     4921 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     4981 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     5041 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     5101 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     5161 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     5221 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     5281 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     5341 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5401 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     5461 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     5521 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     5581 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5641 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5701 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5761 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     5821 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     5881 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     5941 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     6001 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     6061 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     6121 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     6181 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     6241 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     6301 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     6361 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     6421 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     6481 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     6541 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     6601 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     6661 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     6721 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     6781 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     6841 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     6901 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     6961 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     7021 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     7081 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     7141 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     7201 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7261 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7321 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7381 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7441 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7501 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7561 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7621 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7681 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7741 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7801 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7861 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7921 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     7981 ggtcatta
//
