LOCUS       sequence-426497-        6084 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 6084)
  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 6084)
  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: pMK465 (mAB-Hygro)
FEATURES             Location/Qualifiers
     source          1..6084
                     /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"
     CDS             2232..3251
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2232..3251
                     /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    3814..3847
                     /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(3896..3916)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3896..3914)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3935..3951)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3935..3951)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3948..3970)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3959..3975
                     /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(3983..3989,3990..4007,4008..4013))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4028..4049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4166..4183)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4337..4925)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4417..4436)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5096..5887,5888..5956))
                     /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     5719..5738
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5957..6061)
                     /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 tcgagcagct gaagcttacc gctagcatgg
     2221 atagatccgg aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     2281 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     2341 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     2401 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     2461 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     2521 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     2581 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     2641 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     2701 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     2761 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     2821 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     2881 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     2941 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     3001 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     3061 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     3121 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     3181 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     3241 caaaggaata ggctagccgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     3301 gaccccatgc atcgatgata tcagatcccc gggatgcaga aattgatgat ctattaaaca
     3361 ataaagatgt ccactaaaat ggaagttttt cctgtcatac tttgttaaga agggtgagaa
     3421 cagagtacct acattttgaa tggaaggatt ggagctacgg gggtgggggt ggggtgggat
     3481 tagataaatg cctgctcttt actgaaggct ctttactatt gctttatgat aatgtttcat
     3541 agttggatat cataatttaa acaagcaaaa ccaaattaag ggccagctca ttcctcccac
     3601 tcatgatcta tagatctata gatctctcgt gggatcattg tttttctctt gattcccact
     3661 ttgtggttct aagtactgtg gtttccaaat gtgtcagttt catagcctga agaacgagat
     3721 cagcagcctc tgttccacat acacttcatt ctcagtattg ttttgccaag ttctaattcc
     3781 atcagaagct ggtcgagatc cggaaccctt aatataactt cgtataatgt atgctatacg
     3841 aagttattag gtccctcgaa gaggttcact aggatccggt acccagcttt tgttcccttt
     3901 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3961 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4021 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4081 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4141 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4201 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4261 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4321 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4381 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4441 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4501 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4561 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4621 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4681 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4741 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4801 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4861 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4921 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4981 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5041 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5101 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5161 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5221 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5281 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5341 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5401 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5461 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5521 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5581 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5641 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5701 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5761 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5821 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5881 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5941 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6001 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6061 gcacatttcc ccgaaaagtg ccac
//
