LOCUS       sequence-461767-        4475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR mediated KI of BromoTag at N-terminus of 
            BRD2-GFP-P2A-BromoTag, works well with KHC064 and KHC065.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4475)
  AUTHORS   Bond AG, Craigon C, Chan KH, Testa A, Karapetsas A, Fasimoye R, 
            Macartney T, Blow JJ, Alessi DR, Ciulli A
  TITLE     Development of BromoTag: A ""Bump-and-Hole""-PROTAC System to Induce
            Potent, Rapid, and Selective Degradation of Tagged Target Proteins.
  JOURNAL   J Med Chem. 2021 Oct 28;64(20):15477-15502. doi: 
            10.1021/acs.jmedchem.1c01532. Epub 2021 Oct 15.
  PUBMED    34652918
REFERENCE   2  (bases 1 to 4475)
  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: 
            "10.1021/acs.jmedchem.1c01532"; journalName: "J Med Chem"; date: 
            "2021-10-28- 28"; volume: "64"; issue: "20"; pages: "15477-15502"
COMMENT     Sequence Label: KHC068 BRD2-KI-GFP-2A-L387A
FEATURES             Location/Qualifiers
     source          1..4475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     285..307
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     299..316
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     300..316
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     327..346
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        327..345
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             350..361
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             join(878..880,881..883,884..1594)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(878..880,881..883,884..1594)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(923..944)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1184..1203)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1531..1552
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1604..1660
                     /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"
     primer_bind     complement(2688..2705)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2798..3386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2878..2897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3560..4354)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(4355..4459)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
       61 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
      121 agaccgagat agggttgagt ggccgctaca gggcgctccc attcgccatt caggctgcgc
      181 aactgttggg aagggcgttt cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      241 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      301 taaaacgacg gccagtgagc gcgacgtaat acgactcact atagggcgaa ttgaaggaag
      361 gccgtcaagg ccgcatccgc ccaatcctcg gagtctgtcc accccctcta ctccgccctc
      421 aagaggattt caaagatgga ggcggcggct ccctaaacca cttttcgtgt tcatccgcct
      481 ccatccgaga tcgaaacggg acctcgtcgg ccccgtaggg gcccgacaag aagagggaat
      541 ccctgcagac caacagcggg ctatattgac gacggtgtct gagatcgggg accgtctttt
      601 gaagagtcag tccctcctta gttgcccgcc tcagctgagg ccgccgccat tttcttgctg
      661 tccgccgtct gcagagcgcg ccaagctgcc cggagctctc cgagaggccc caaagagact
      721 gctttcgtgc cggccaggca gggggtttgt cgcctggagg cccaagagga acggcctccc
      781 cccaacttag cgggttatgc tggaccgggc ggtgagggga accgagggca cccggacttt
      841 ccgcggctga gggcagcgcc ggttcgttgc ggtcaagatg gtgagcaagg gcgaggagct
      901 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      961 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1021 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1081 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1141 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1201 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1261 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1321 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1381 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1441 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1501 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1561 cgggatcact ctcggcatgg acgagctgta caagggatcc ggagcaacaa acttctcact
     1621 actcaaacaa gcaggtgacg tggaggagaa tcccgggcct gggtccgtga aggacgtgcc
     1681 cgactctcag cagcacccag caccagagaa gagcagcaag gtctcggagc agctcaagtg
     1741 ctgcagcggc atcctcaagg agatgtttgc caagaagcac gccgcctacg cctggccctt
     1801 ctacaagcct gtggacgtgg aggcactggg cgcccacgac tactgtgaca tcatcaagca
     1861 ccccatggac atgagcacaa tcaagtctaa actggaggcc cgtgagtacc gtgatgctca
     1921 ggagtttggt gctgacgtcc gattgatgtt ctccaactgc tataagtaca accctcctga
     1981 ccatgaggtg gtggccatgg cccgcaagct ccaggatgtg ttcgaaatgc gctttgccaa
     2041 gatgccggac gagggcggag gcggaggtgg cctgcaaaac gtgactcccc acaataagta
     2101 cgtttccgcg agccgcgtct gggaagggga tgttgcaggg cggcggcaca ggggtgtggg
     2161 gcgccgtgtt gggagtactg agcggccccg gcgcgctgct gttgcggcgc agctgtcgac
     2221 tcggtcgcgc ggagggaatt gagcgacggt tttggaacgg tggtggcggc tcggctactg
     2281 ctcgtggagg ggaatacagg ttgtcaattt atacgctatt aatgccgccg tggcccagtc
     2341 ttaaccgagt caggcagagc tagtttgacg gtggagtgga gtgaggttga acagcaggtt
     2401 tggcgtttgg tgggtctggt atctagcggc ggtctgttag ccttttaggg gggattcacg
     2461 gacacctcta gcgccctgta gggttgccat ggtgacggag cgcttaaggg actggcaacg
     2521 gggattccca gagaagggta aagggatcac tctcccgtgt gtgcaggttc ctactgggcc
     2581 tcatgggcct tcctttcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2641 taacatggtc atagctgttt ccttgcgtat tgggcgctct ccgcttcctc gctcactgac
     2701 tcgctgcgct cggtcgttcg ggtaaagcct ggggtgccta atgagcaaaa ggccagcaaa
     2761 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2821 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2881 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2941 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3001 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3061 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3121 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3181 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3241 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3301 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3361 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3421 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3481 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3541 aaacttggtc tgacagttat tagaaaaatt catccagcag acgataaaac gcaatacgct
     3601 ggctatccgg tgccgcaatg ccatacagca ccagaaaacg atccgcccat tcgccgccca
     3661 gttcttccgc aatatcacgg gtggccagcg caatatcctg ataacgatcc gccacgccca
     3721 gacggccgca atcaataaag ccgctaaaac ggccattttc caccataatg ttcggcaggc
     3781 acgcatcacc atgggtcacc accagatctt cgccatccgg catgctcgct ttcagacgcg
     3841 caaacagctc tgccggtgcc aggccctgat gttcttcatc cagatcatcc tgatccacca
     3901 ggcccgcttc catacgggta cgcgcacgtt caatacgatg tttcgcctga tgatcaaacg
     3961 gacaggtcgc cgggtccagg gtatgcagac gacgcatggc atccgccata atgctcactt
     4021 tttctgccgg cgccagatgg ctagacagca gatcctgacc cggcacttcg cccagcagca
     4081 gccaatcacg gcccgcttcg gtcaccacat ccagcaccgc cgcacacgga acaccggtgg
     4141 tggccagcca gctcagacgc gccgcttcat cctgcagctc gttcagcgca ccgctcagat
     4201 cggttttcac aaacagcacc ggacgaccct gcgcgctcag acgaaacacc gccgcatcag
     4261 agcagccaat ggtctgctgc gcccaatcat agccaaacag acgttccacc cacgctgccg
     4321 ggctacccgc atgcaggcca tcctgttcaa tcatactctt cctttttcaa tattattgaa
     4381 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4441 aacaaatagg ggttccgcgc acatttcccc gaaaa
//
