LOCUS       sequence-221658-        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-3A
FEATURES             Location/Qualifiers
     source          1..5596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..421
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        422..625
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     576..596
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      664..673
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(670..672,673..675,676..1386)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(715..736)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(976..995)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1323..1344
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1432..1452
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1465..1509
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    2441..2562
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2478..2497)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2532..2551
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2569..3024)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2706..2727)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2918..2937
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3051..3155
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3157..3514
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3157..3348
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3180..3200)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3365..3500
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3549..4343
                     /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             3549..4343
                     /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(3603..3622)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4213..4232
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4531..4550)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4575..4622
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4951..5539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5440..5459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcattag
       61 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      121 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      181 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      241 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      301 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      361 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      421 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      481 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      541 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      601 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg
      661 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      721 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      781 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      841 tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac
      901 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      961 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1021 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1081 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1141 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1201 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1261 gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1321 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1381 tacaagtccg gaggggatcg gtggagctcc accggtggcg gccgctctag agagaatctt
     1441 tattttcagg gcgccgccaa attcaaagaa accgctgctg ctaaattcga acgccagcac
     1501 atggacagcg gaggtggagg tactagatct gtggatccaa tgacctacga ggcccagttc
     1561 tttggcttca cgccacaaac gtgcatgctt cggatctaca ttgcatttca agactaccta
     1621 tttgaagtga tgcaggccgt tgaacaggtt attctgaaga agctggatgg catcccagac
     1681 tgtgacatta gcccagtgca gattcgcaaa tgcacagaga agtttctttg cttcatgaaa
     1741 ggacattttg ataacctttt tagcaaaatg gagcaactgt ttttgcagct gattttacgt
     1801 attccctcaa acatcttgct tcctgaagat aaatgtaagg agacacctta tagtgaggaa
     1861 gattttcagc atctccagaa agaaattgaa cagttacagg agaagtacaa gactgaatta
     1921 tgtactaagc aggcccttct tgcagaatta gaagagcaaa aaattgttca ggccaaactc
     1981 aaacagacgt tgactttctt tgatgagctt cataatgttg gcagagatca tgggactagt
     2041 gattttaggg agagtttagt atccctggtt cagaactcca gaaaactaca gaacattaga
     2101 gacaatgtgg aaaaggaatc gaaacgactg aaaatatctt ctagtctcga caagacctct
     2161 gaggaccagc agacagcttg tggcactatc tacagtcaga ctctcagcat caagaagctg
     2221 gccccaatta ttgaagacgc tcgtgaagcc gcacactcct ctggcttctc tggttcttct
     2281 gcctcggttg caagcacctc ctcctgactg aggatccacc ggatctagat aactgatcat
     2341 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     2401 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2461 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2521 gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg
     2581 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     2641 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     2701 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     2761 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     2821 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2881 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2941 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     3001 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3061 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3121 gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg
     3181 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3241 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3301 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3361 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3421 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3481 aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat
     3541 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3601 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3661 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3721 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3781 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3841 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3901 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3961 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4021 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     4081 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4141 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4201 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4261 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4321 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4381 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4441 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4501 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg
     4561 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt
     4621 tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
     4681 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga
     4741 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc
     4801 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4861 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4921 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4981 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5041 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5101 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5161 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5221 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5281 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5341 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5401 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5461 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5521 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5581 ctggcctttt gctcac
//
