LOCUS       sequence-426341-        5037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BromoTag for C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5037)
  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 5037)
  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: pMK448 (BromoTag-BSD)
FEATURES             Location/Qualifiers
     source          1..5037
                     /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"
     polyA_signal    1091..1212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1128..1147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1182..1201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1243..1276
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1337..1836
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1372..1395)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1769..1788
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1860..1869
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1866..2264
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    2767..2800
                     /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(2849..2869)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2849..2867)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2888..2904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2888..2904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2901..2923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2912..2928
                     /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(2936..2942,2943..2960,2961..2966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2981..3002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3119..3136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3290..3878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3370..3389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4049..4840,4841..4909))
                     /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     4672..4691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4910..5014)
                     /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 gggtccgtga aggacgtgcc cgactctcag cagcacccag
      721 caccagagaa gagcagcaag gtctcggagc agctcaagtg ctgcagcggc atcctcaagg
      781 agatgtttgc caagaagcac gccgcctacg cctggccctt ctacaagcct gtggacgtgg
      841 aggcactggg cgcccacgac tactgtgaca tcatcaagca ccccatggac atgagcacaa
      901 tcaagtctaa actggaggcc cgtgagtacc gtgatgctca ggagtttggt gctgacgtcc
      961 gattgatgtt ctccaactgc tataagtaca accctcctga ccatgaggtg gtggccatgg
     1021 cccgcaagct ccaggatgtg ttcgaaatgc gctttgccaa gatgccggac gagtaacaat
     1081 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1201 caatgtatct taacgcgtcg atcatattca ataaccctta atataacttc gtataatgta
     1261 tgctatacga agttattagg tctgaagagg agtttacgtc cagccaagct taggatctcg
     1321 acctcgaaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     1381 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     1441 acatccaccg gtaggcgcca accgactccg ttctttggtg gccccttcgc gccaccttct
     1501 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     1561 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     1621 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     1681 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
     1741 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
     1801 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcatccatct agatctcgag
     1861 ccaccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     1921 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     1981 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     2041 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     2101 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     2161 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     2221 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaatcgatg atatcagatc
     2281 cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa aatggaagtt
     2341 tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt gaatggaagg
     2401 attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc tttactgaag
     2461 gctctttact attgctttat gataatgttt catagttgga tatcataatt taaacaagca
     2521 aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct atagatctct
     2581 cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact gtggtttcca
     2641 aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca catacacttc
     2701 attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag atccggaacc
     2761 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc gaagaggttc
     2821 actaggatcc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     2881 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2941 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3001 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3061 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3121 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3181 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3241 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3301 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3361 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3421 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3481 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3541 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3601 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3661 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3721 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3781 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3841 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3901 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3961 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4021 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4081 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4141 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4201 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4261 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4321 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4381 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4441 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4501 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4561 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4621 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4681 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4741 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4801 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4861 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4921 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4981 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
