LOCUS       sequence-461769-        5416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KHC065 (pBABE pU6 BRD2 K1-2C), gRNA and cas9D10A mammalian 
            expression vector for CRISPR mediated cutting of BRD2 at the 
            N-terminus, use with KHC064.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5416)
  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 5416)
  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: KHC065 pBABE pU6 BRD2 K1-2C
FEATURES             Location/Qualifiers
     source          1..5416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    523..722
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             923..1339
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1269..1291
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1307..1323
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_RNA        complement(1370..1445)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1467..1715)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1525..1544)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1695..1715)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1727..1747)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1732..2061
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1912..2047
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2071..2670
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2071..2090)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2567..2587
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2891..3360
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3490..3507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3661..4249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3741..3760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4409..5200,5201..5269))
                     /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     5032..5051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5270..5374)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             join(5405..5416,1..458)
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
ORIGIN
        1 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggaa
       61 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
      121 caagaacaga tggtccccag atgcggtcca gccctcagca gtttctagag aaccatcaga
      181 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
      241 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
      301 aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
      361 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
      421 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
      481 ggagacccct gcccagggac caccgaccca ccaccgggag gcaagctggc cagcaactta
      541 tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
      601 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tctgaacacc
      661 cggccgcaac cctgggagac gtcccaggga ctttgggggc cgtttttgtg gcccgacctg
      721 aggaagggag tcgatgtgga atccgacccc gtcaggatat gtggttctgg taggagacga
      781 gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttgga accgaagccg
      841 cgcgtcttgt ctgctgcagc gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt
      901 tctgtatttg tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg
      961 tcactggaaa gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg
     1021 ttgggttacc ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg
     1081 cacctttaac cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca
     1141 tggacaccca gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc
     1201 tccctgggtc aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc
     1261 gtctctcccc cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct
     1321 cactccttct ctaggcgccg gccggatctg cggccgcgaa ttcaaaaaag caccgactcg
     1381 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat ttctagctct
     1441 aaaaccccgg actttccgcg gctgacggtg tttcgtcctt tccacaagat atataaagcc
     1501 aagaaatcga aatactttca agttacggta agcatatgat agtccatttt aaaacataat
     1561 tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat
     1621 atctttgtgt ttacagtcaa attaattcta attatctctc taacagcctt gtatcgtata
     1681 tgcaaatatg aaggaatcat gggaaatagg ccctcgtcga ccctgtggaa tgtgtgtcag
     1741 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1801 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1861 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     1921 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     1981 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     2041 ggaggcctag gcttttgcaa aaagcttacc atgaccgagt acaagcccac ggtgcgcctc
     2101 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     2161 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     2221 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     2281 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     2341 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     2401 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     2461 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     2521 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     2581 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     2641 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     2701 gaaaggagcg cacgacccca tgcatcgata aaataaaaga ttttatttag tctccagaaa
     2761 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt
     2821 tgcaaggcat ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca
     2881 gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     2941 tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     3001 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag
     3061 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
     3121 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
     3181 cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga
     3241 gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct
     3301 cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca
     3361 catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc
     3421 atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3481 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3541 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3601 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3661 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3721 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3781 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3841 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3901 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3961 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4021 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4081 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4141 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4201 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4261 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4321 catgagatta tcaaaaagga tcttcaccta gatcagtttg cggccgtaaa tcaatctaaa
     4381 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4441 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4501 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4561 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4621 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4681 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4741 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4801 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4861 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4921 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4981 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5041 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5101 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5161 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5221 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5281 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5341 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5401 cagcctgaat atgggc
//
