LOCUS       sequence-133563-        5051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mAID using CRISPR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5051)
  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 5051)
  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: pMK288 (mAID-Bsr)
FEATURES             Location/Qualifiers
     source          1..5051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             681..884
                     /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"
     polyA_signal    1005..1126
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1042..1061)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1096..1115
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1157..1190
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1251..1750
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1286..1309)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1683..1702
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    2781..2814
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2863..2883)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2863..2881)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2902..2918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2902..2918)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2915..2937)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2926..2942
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2950..2956,2957..2974,2975..2980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2995..3016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3133..3150)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3304..3892)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3384..3403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4063..4854,4855..4923))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4686..4705
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4924..5028)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctccc
      661 cgggatccgg tgcaggcgcc aaggagaaga gtgcttgtcc taaagatcca gccaaacctc
      721 cggccaaggc acaagttgtg ggatggccac cggtgagatc ataccggaag aacgtgatgg
      781 tttcctgcca aaaatcaagc ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg
      841 acggagcacc gtacttgagg aaaatcgatt tgaggatgta taaataatct agataactga
      901 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
      961 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1021 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1081 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtcgatcata
     1141 ttcaataacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctgaa
     1201 gaggagttta cgtccagcca agcttaggat ctcgacctcg aaattctacc gggtagggga
     1261 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
     1321 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccgac
     1381 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
     1441 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
     1501 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
     1561 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
     1621 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
     1681 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
     1741 gggcctttcg acctgcatcc atctagatct cgagcagctg aagcttaccg ctagcatgaa
     1801 aacatttaac atttctcaac aagatctaga attagtagaa gtagcgacag agaagattac
     1861 aatgctttat gaggataata aacatcatgt gggagcggca attcgtacga aaacaggaga
     1921 aatcatttcg gcagtacata ttgaagcgta tataggacga gtaactgttt gtgcagaagc
     1981 cattgcgatt ggtagtgcag tttcgaatgg acaaaaggat tttgacacga ttgtagctgt
     2041 tagacaccct tattctgacg aagtagatag aagtattcga gtggtaagtc cttgtggtat
     2101 gtgtagggag ttgatttcag actatgcacc agattgtttt gtgttaatag aaatgaatgg
     2161 caagttagtc aaaactacga ttgaagaact cattccactc aaatataccc gaaattaagc
     2221 tagccgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
     2281 gatgatatca gatccccggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca
     2341 ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca
     2401 ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct
     2461 gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat
     2521 aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag
     2581 atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag
     2641 tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt
     2701 tccacataca cttcattctc agtattgttt tgccaagttc taattccatc agaagctggt
     2761 cgagatccgg aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc
     2821 cctcgaagag gttcactagg atccggtacc cagcttttgt tccctttagt gagggttaat
     2881 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2941 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3001 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3061 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3121 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3181 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3241 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3301 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3361 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3421 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3481 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3541 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3601 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3661 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3721 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3781 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3841 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3901 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3961 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4021 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4081 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4141 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4201 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4261 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4321 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4381 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4441 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4501 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4561 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4621 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4681 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4741 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4801 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4861 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4921 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4981 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5041 aaaagtgcca c
//
