LOCUS       sequence-426500-        5372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mAID-BromoTag for C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5372)
  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 5372)
  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: pMK466 (mAB-BSD)
FEATURES             Location/Qualifiers
     source          1..5372
                     /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"
     polyA_signal    1426..1547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1463..1482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1517..1536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1578..1611
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1672..2171
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1707..1730)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2104..2123
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2195..2204
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2201..2599
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    3102..3135
                     /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(3184..3204)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3184..3202)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3223..3239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3223..3239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3236..3258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3247..3263
                     /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(3271..3277,3278..3295,3296..3301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3316..3337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3454..3471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3625..4213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3705..3724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4384..5175,5176..5244))
                     /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     5007..5026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5245..5349)
                     /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 cggatcagtg aaggacgtgc ccgactctca gcagcaccca gcaccagaga agagcagcaa
      961 ggtctcggag cagctcaagt gctgcagcgg catcctcaag gagatgtttg ccaagaagca
     1021 cgccgcctac gcctggccct tctacaagcc tgtggacgtg gaggcactgg gcgcccacga
     1081 ctactgtgac atcatcaagc accccatgga catgagcaca atcaagtcta aactggaggc
     1141 ccgtgagtac cgtgatgctc aggagtttgg tgctgacgtc cgattgatgt tctccaactg
     1201 ctataagtac aaccctcctg accatgaggt ggtggccatg gcccgcaagc tccaggatgt
     1261 gttcgaaatg cgctttgcca agatgccgga cgagtagtgt acaagtaatc tagataactg
     1321 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     1381 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     1441 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1501 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtcgatcat
     1561 attcaataac ccttaatata acttcgtata atgtatgcta tacgaagtta ttaggtctga
     1621 agaggagttt acgtccagcc aagcttagga tctcgacctc gaaattctac cgggtagggg
     1681 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     1741 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccga
     1801 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     1861 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     1921 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     1981 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     2041 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     2101 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     2161 cgggcctttc gacctgcatc catctagatc tcgagccacc atggccaagc ctttgtctca
     2221 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
     2281 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
     2341 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
     2401 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
     2461 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
     2521 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
     2581 ttatgtgtgg gagggctaat cgatgatatc agatccccgg gatgcagaaa ttgatgatct
     2641 attaaacaat aaagatgtcc actaaaatgg aagtttttcc tgtcatactt tgttaagaag
     2701 ggtgagaaca gagtacctac attttgaatg gaaggattgg agctacgggg gtgggggtgg
     2761 ggtgggatta gataaatgcc tgctctttac tgaaggctct ttactattgc tttatgataa
     2821 tgtttcatag ttggatatca taatttaaac aagcaaaacc aaattaaggg ccagctcatt
     2881 cctcccactc atgatctata gatctataga tctctcgtgg gatcattgtt tttctcttga
     2941 ttcccacttt gtggttctaa gtactgtggt ttccaaatgt gtcagtttca tagcctgaag
     3001 aacgagatca gcagcctctg ttccacatac acttcattct cagtattgtt ttgccaagtt
     3061 ctaattccat cagaagctgg tcgagatccg gaacccttaa tataacttcg tataatgtat
     3121 gctatacgaa gttattaggt ccctcgaaga ggttcactag gatccggtac ccagcttttg
     3181 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     3241 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3301 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3361 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3421 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3481 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3541 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3601 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3661 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3721 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3781 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3841 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3901 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3961 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4021 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4081 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4141 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4201 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4261 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4321 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4381 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4441 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4501 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4561 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4621 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4681 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4741 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4801 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4861 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4921 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4981 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5041 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5101 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5161 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5221 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5281 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5341 ggttccgcgc acatttcccc gaaaagtgcc ac
//
