LOCUS       sequence-426506-        6018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mAID-dTAG for C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6018)
  AUTHORS   Hatoyama Y, Islam M, Bond AG, Hayashi KI, Ciulli A, Kanemaki MT
  TITLE     Combination of AID2 and BromoTag expands the utility of degron-based
            protein knockdowns.
  JOURNAL   EMBO Rep. 2024 Sep;25(9):4062-4077. doi: 10.1038/s44319-024-00224-4.
            Epub 2024 Aug 23.
  PUBMED    39179892
REFERENCE   2  (bases 1 to 6018)
  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: 
            "10.1038/s44319-024-00224-4"; journalName: "EMBO Rep"; date: 
            "2024-09"; volume: "25"; issue: "9"; pages: "4062-4077"
COMMENT     Sequence Label: pMK469 (mAID-dTAG BSD)
FEATURES             Location/Qualifiers
     source          1..6018
                     /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             680..883
                     /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             908..1231
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    1360..1481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1397..1416)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1451..1470
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1512..1545
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1606..2105
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1641..1664)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2038..2057
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2166..3185
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2166..3185
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     protein_bind    3748..3781
                     /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(3830..3850)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3830..3848)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3869..3885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3869..3885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3882..3904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3893..3909
                     /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(3917..3923,3924..3941,3942..3947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3962..3983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4100..4117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4271..4859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4351..4370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5030..5821,5822..5890))
                     /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     5653..5672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5891..5995)
                     /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 tggagctcgg
      661 atccggtgca ggcgcctcta aggagaagag tgcttgtcct aaagatccag ccaaacctcc
      721 ggccaaggca caagttgtgg gatggccacc ggtgagatca taccggaaga acgtgatggt
      781 ttcctgccaa aaatcaagcg gtggcccgga ggcggcggcg ttcgtgaagg tatcaatgga
      841 cggagcaccg tacttgagga aaatcgattt gaggatgtat aaaggcgctg gtgcaggcgc
      901 cggatcagga gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg
      961 cggccagacc tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aagttgattc
     1021 ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg
     1081 ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc
     1141 agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt
     1201 cttcgatgtg gagcttctaa aactggaata gtgtacaagt aatctagata actgatcata
     1261 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     1321 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     1381 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1441 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtcga tcatattcaa
     1501 taacccttaa tataacttcg tataatgtat gctatacgaa gttattaggt ctgaagagga
     1561 gtttacgtcc agccaagctt aggatctcga cctcgaaatt ctaccgggta ggggaggcgc
     1621 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     1681 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccgactccgt
     1741 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     1801 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     1861 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     1921 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     1981 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     2041 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     2101 tttcgacctg catccatcta gatctcgagc agctgaagct taccgctagc atggatagat
     2161 ccggaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     2221 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     2281 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     2341 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     2401 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     2461 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     2521 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     2581 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     2641 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     2701 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     2761 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     2821 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     2881 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     2941 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     3001 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     3061 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     3121 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     3181 aataggctag ccgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc
     3241 atgcatcgat gatatcagat ccccgggatg cagaaattga tgatctatta aacaataaag
     3301 atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg agaacagagt
     3361 acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg ggattagata
     3421 aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt tcatagttgg
     3481 atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc ccactcatga
     3541 tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc cactttgtgg
     3601 ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg agatcagcag
     3661 cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa ttccatcaga
     3721 agctggtcga gatccggaac ccttaatata acttcgtata atgtatgcta tacgaagtta
     3781 ttaggtccct cgaagaggtt cactaggatc cggtacccag cttttgttcc ctttagtgag
     3841 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3901 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3961 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4021 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4081 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4141 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4201 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4261 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4321 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4381 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4441 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4501 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4561 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4621 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4681 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4741 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4801 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4861 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4921 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4981 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5041 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5101 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5161 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5221 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5281 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5341 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5401 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5461 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5521 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5581 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5641 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5701 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5761 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5821 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5881 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5941 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6001 ttccccgaaa agtgccac
//
