LOCUS       sequence-426342-        5881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BromoTag-mClover for C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5881)
  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 5881)
  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: pMK449 (BromoTag-mClover-BSD)
FEATURES             Location/Qualifiers
     source          1..5881
                     /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             join(1098..1100,1101..1103,1104..1817)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1143..1164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1751..1772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1935..2056
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1972..1991)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2026..2045
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2087..2120
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2181..2680
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2216..2239)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2613..2632
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2704..2713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2710..3108
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    3611..3644
                     /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(3693..3713)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3693..3711)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3732..3748)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3732..3748)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3745..3767)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3756..3772
                     /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(3780..3786,3787..3804,3805..3810))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3825..3846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3963..3980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4134..4722)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4214..4233)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4893..5684,5685..5753))
                     /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     5516..5535
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5754..5858)
                     /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 gagggcggag
     1081 gcggaggtgg cgctagcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1141 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg
     1201 agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1261 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc ttcagccgct
     1321 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1381 aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt
     1441 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1501 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcacgg
     1561 ccgacaagca gaagaacggc atcaaggcta acttcaagat ccgccacaac gttgaggacg
     1621 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1681 tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac cccaacgaga
     1741 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggattaca catggcatgg
     1801 acgagctgta caagtaatct agataactga tcataatcag ccataccaca tttgtagagg
     1861 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     1921 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1981 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2041 tcatcaatgt atcttaacgc gtcgatcata ttcaataacc cttaatataa cttcgtataa
     2101 tgtatgctat acgaagttat taggtctgaa gaggagttta cgtccagcca agcttaggat
     2161 ctcgacctcg aaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     2221 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     2281 ttccacatcc accggtaggc gccaaccgac tccgttcttt ggtggcccct tcgcgccacc
     2341 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     2401 gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca
     2461 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     2521 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
     2581 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     2641 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcatcc atctagatct
     2701 cgagccacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     2761 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     2821 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     2881 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     2941 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     3001 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     3061 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaatc gatgatatca
     3121 gatccccggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca ctaaaatgga
     3181 agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca ttttgaatgg
     3241 aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct gctctttact
     3301 gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat aatttaaaca
     3361 agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag atctatagat
     3421 ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag tactgtggtt
     3481 tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt tccacataca
     3541 cttcattctc agtattgttt tgccaagttc taattccatc agaagctggt cgagatccgg
     3601 aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc cctcgaagag
     3661 gttcactagg atccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     3721 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3781 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3841 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3901 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3961 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4021 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4081 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4141 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4201 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4261 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4321 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4381 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4441 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4501 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4561 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4621 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4681 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4741 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4801 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4861 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4921 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4981 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5041 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5101 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5161 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5221 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5281 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5341 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5401 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5461 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5521 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5581 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5641 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5701 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5761 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5821 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5881 c
//
