LOCUS       sequence-138286-        1051 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  C-terminal tagging with mAID-mClover using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1051)
  AUTHORS   Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
  TITLE     Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron 
            Tagging with Short Homology Donors.
  JOURNAL   Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001.
            Epub 2016 Mar 24.
  PUBMED    27052166
REFERENCE   2  (bases 1 to 1051)
  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.celrep.2016.03.001"; journalName: "Cell Rep"; date: 
            "2016-04-5- 5"; volume: "15"; issue: "1"; pages: "210-8"
COMMENT     Sequence Label: pMK289 (mAID-mClover-NeoR)
FEATURES             Location/Qualifiers
     source          1..1051
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             62..265
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(272..274,275..277,278..991)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(317..338)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     925..946
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 cgcgcgtaat acgactcact atagggcgaa ttggagctcc ccgggatccg gtgcaggcgc
       61 caaggagaag agtgcttgtc ctaaagatcc agccaaacct ccggccaagg cacaagttgt
      121 gggatggcca ccggtgagat cataccggaa gaacgtgatg gtttcctgcc aaaaatcaag
      181 cggtggcccg gaggcggcgg cgttcgtgaa ggtatcaatg gacggagcac cgtacttgag
      241 gaaaatcgat ttgaggatgt ataaagctag catggtgagc aagggcgagg agctgttcac
      301 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      361 ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
      421 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcgtggc
      481 ctgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      541 cgaaggctac gtccaggagc gcaccatctc tttcaaggac gacggtacct acaagacccg
      601 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
      661 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa
      721 cgtctatatc acggccgaca agcagaagaa cggcatcaag gctaacttca agatccgcca
      781 caacgttgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
      841 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc catcagtcca agctgagcaa
      901 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
      961 tacacatggc atggacgagc tgtacaagta atctagataa ctgatcataa tcagccatac
     1021 cacatttgta gaggttttac ttgctttaaa a
//
