LOCUS       sequence-221655-        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
FEATURES             Location/Qualifiers
     source          1..5596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        89..392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        393..596
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     547..567
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      635..644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(641..643,644..646,647..1357)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(686..707)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(947..966)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1294..1315
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1403..1423
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1436..1480
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    2412..2533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2449..2468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2503..2522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2540..2995)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2677..2698)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2889..2908
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3022..3126
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3128..3485
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3128..3319
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3151..3171)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3336..3471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3520..4314
                     /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             3520..4314
                     /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(3574..3593)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4184..4203
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4502..4521)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4546..4593
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4922..5510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5411..5430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat
       61 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      121 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      181 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      241 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      301 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      361 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      421 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      481 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      541 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt
      601 tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc atggtgagca agggcgagga
      661 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      721 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      781 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta
      841 cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      901 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
      961 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1021 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1081 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1141 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1201 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc
     1261 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1321 cgccgggatc actctcggca tggacgagct gtacaagtcc ggaggggatc ggtggagctc
     1381 caccggtggc ggccgctcta gagagaatct ttattttcag ggcgccgcca aattcaaaga
     1441 aaccgctgct gctaaattcg aacgccagca catggacagc ggaggtggag gtactagatc
     1501 tgtggatcca atgacctacg aggcccagtt ctttggcttc acgccacaaa cgtgcatgct
     1561 tcggatctac attgcatttc aagactacct atttgaagtg atgcaggccg ttgaacaggt
     1621 tattctgaag aagctggatg gcatcccaga ctgtgacatt agcccagtgc agattcgcaa
     1681 atgcacagag aagtttcttt gcttcatgaa aggacatttt gataaccttt ttagcaaaat
     1741 ggagcaactg tttttgcagc tgattttacg tattccctca aacatcttgc ttcctgaaga
     1801 taaatgtaag gagacacctt atagtgagga agattttcag catctccaga aagaaattga
     1861 acagttacag gagaagtaca agactgaatt atgtactaag caggcccttc ttgcagaatt
     1921 agaagagcaa aaaattgttc aggccaaact caaacagacg ttgactttct ttgatgagct
     1981 tcataatgtt ggcagagatc atgggactag tgattttagg gagagtttag tatccctggt
     2041 tcagaactcc agaaaactac agaacattag agacaatgtg gaaaaggaat cgaaacgact
     2101 gaaaatatct tctagtctcg acaagacctc tgaggaccag cagacagctt gtggcactat
     2161 ctacagtcag actctcagca tcaagaagct gagcccaatt attgaagaca gtcgtgaagc
     2221 cacacactcc tctggcttct ctggttcttc tgcctcggtt gcaagcacct cctcctgact
     2281 gaggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     2341 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2401 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2461 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2521 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2581 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2641 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2701 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2761 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2821 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2881 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2941 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3001 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3061 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3121 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3241 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3301 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3361 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3421 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3481 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3541 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3601 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3661 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3721 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3781 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3841 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3901 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3961 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4021 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4081 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4141 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4201 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4261 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4321 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4381 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4441 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4501 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4561 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     4621 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     4681 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4741 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     4801 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4861 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4921 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4981 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5041 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5101 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5161 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5221 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5281 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5341 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5401 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5461 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5521 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5581 cgttatcccc tgattc
//
