LOCUS       sequence-221659-        5596 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 5596)
  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 5596)
  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-MIS12-KARD-3D
FEATURES             Location/Qualifiers
     source          1..5596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        17..320
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        321..524
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     475..495
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      563..572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(569..571,572..574,575..1285)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(614..635)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(875..894)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1222..1243
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1331..1351
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1364..1408
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    2340..2461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2377..2396)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2431..2450
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2468..2923)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2605..2626)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2817..2836
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2950..3054
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3056..3413
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3056..3247
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3079..3099)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3264..3399
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3448..4242
                     /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             3448..4242
                     /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(3502..3521)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4112..4131
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4430..4449)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4474..4521
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4850..5438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5339..5358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
       61 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      121 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      181 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      241 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      301 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      361 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      421 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      481 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      541 agatccgcta gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      601 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      661 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      721 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg
      781 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      841 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      901 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      961 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1021 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1081 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1141 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga
     1201 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1261 tctcggcatg gacgagctgt acaagtccgg aggggatcgg tggagctcca ccggtggcgg
     1321 ccgctctaga gagaatcttt attttcaggg cgccgccaaa ttcaaagaaa ccgctgctgc
     1381 taaattcgaa cgccagcaca tggacagcgg aggtggaggt actagatctg tggatccaat
     1441 gacctacgag gcccagttct ttggcttcac gccacaaacg tgcatgcttc ggatctacat
     1501 tgcatttcaa gactacctat ttgaagtgat gcaggccgtt gaacaggtta ttctgaagaa
     1561 gctggatggc atcccagact gtgacattag cccagtgcag attcgcaaat gcacagagaa
     1621 gtttctttgc ttcatgaaag gacattttga taaccttttt agcaaaatgg agcaactgtt
     1681 tttgcagctg attttacgta ttccctcaaa catcttgctt cctgaagata aatgtaagga
     1741 gacaccttat agtgaggaag attttcagca tctccagaaa gaaattgaac agttacagga
     1801 gaagtacaag actgaattat gtactaagca ggcccttctt gcagaattag aagagcaaaa
     1861 aattgttcag gccaaactca aacagacgtt gactttcttt gatgagcttc ataatgttgg
     1921 cagagatcat gggactagtg attttaggga gagtttagta tccctggttc agaactccag
     1981 aaaactacag aacattagag acaatgtgga aaaggaatcg aaacgactga aaatatcttc
     2041 tagtctcgac aagacctctg aggaccagca gacagcttgt ggcactatct acagtcagac
     2101 tctcagcatc aagaagctgg acccaattat tgaagacgat cgtgaagccg accactcctc
     2161 tggcttctct ggttcttctg cctcggttgc aagcacctcc tcctgactga ggatccaccg
     2221 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2281 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2341 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2401 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2461 aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     2521 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     2581 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     2641 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     2701 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     2761 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2821 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     2881 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     2941 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3001 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     3061 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3121 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3241 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3301 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3361 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     3421 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3481 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3541 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3601 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     3661 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3721 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3781 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3841 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3901 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3961 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4021 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4081 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4141 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4201 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4261 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4321 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4381 gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga
     4441 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     4501 acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     4561 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     4621 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     4681 ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4741 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4801 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4861 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4921 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4981 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5041 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5101 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5161 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5221 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5281 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5341 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5401 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5461 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5521 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
     5581 tcattagttc atagcc
//
