LOCUS       sequence-426340-        4558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BromoTag-mCherry for N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4558)
  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 4558)
  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: pMK447 (BSD-P2A-BromoTag-mCherry)
FEATURES             Location/Qualifiers
     source          1..4558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..460
                     /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     586..608
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     600..617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     601..617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     627..646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        627..645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     670..686
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     671..687
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      702..711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             708..1103
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1116..1172
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1590..2297
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1735..1753)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2317..2333)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2317..2333)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2370..2390)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2370..2388)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2409..2425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2409..2425)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2422..2444)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2433..2449
                     /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(2457..2463,2464..2481,2482..2487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2502..2523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2640..2657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2811..3399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2891..2910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3570..4361,4362..4430))
                     /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     4193..4212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4431..4535)
                     /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 atgagctgat ttaacaaaaa
      421 tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc
      481 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      541 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      601 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
      661 ggccccccct cgaggtcgac ggtatcgata agcttgatat cgccaccatg gccaagcctt
      721 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
      781 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
      841 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
      901 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
      961 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     1021 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     1081 cctctggtta tgtgtgggag ggctcaggaa gcggagctac taacttcagc ctgctgaagc
     1141 aggctggaga cgtggaggag aaccctggac ctagatctgt gaaggacgtg cccgactctc
     1201 agcagcaccc agcaccagag aagagcagca aggtctcgga gcagctcaag tgctgcagcg
     1261 gcatcctcaa ggagatgttt gccaagaagc acgccgccta cgcctggccc ttctacaagc
     1321 ctgtggacgt ggaggcactg ggcgcccacg actactgtga catcatcaag caccccatgg
     1381 acatgagcac aatcaagtct aaactggagg cccgtgagta ccgtgatgct caggagtttg
     1441 gtgctgacgt ccgattgatg ttctccaact gctataagta caaccctcct gaccatgagg
     1501 tggtggccat ggcccgcaag ctccaggatg tgttcgaaat gcgctttgcc aagatgccgg
     1561 acgagggcgg aggcggaggt ggcgctagca tggtgagcaa gggcgaggag gataacatgg
     1621 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     1681 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     1741 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     1801 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     1861 ccttccccga gggcttcaat tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     1921 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     1981 gcaccaactt cccctccgac ggccccgtaa tgcagtgtcg taccatgggc tgggaggcct
     2041 ccactgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     2101 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     2161 ccgtgcagct gcccggcgcc tacaacgtcg acatcaagtt ggacatcctt tcccacaacg
     2221 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     2281 tggacgagct gtacaagggc gctggtgcag gcgccggatc cactagttct agagcggccg
     2341 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     2401 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2461 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2521 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2581 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2641 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2701 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2761 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2821 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2881 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2941 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3001 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3061 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3241 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3661 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3721 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3781 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3841 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3901 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3961 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4021 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4081 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4141 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4201 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4261 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4321 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4381 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4441 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4501 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
