LOCUS       sequence-461799-        4585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid to tag human BRD4 with Halo tag; for 
            CRISPR-mediated gene insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4585)
  TITLE     Imielinski Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 4585)
  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"
COMMENT     Sequence Label: pUC57-Homology Arms-BRD4-HaloTag
FEATURES             Location/Qualifiers
     source          1..4585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     365..382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    499..520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(535..540,541..558,559..565)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    573..589
                     /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)."
     primer_bind     578..600
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     597..613
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     597..613
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1151..1174
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1184..2074
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             2087..2107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(2627..2644)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2627..2643)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2636..2658)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2852..2871)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2971..2993
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3031..3049)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3117..3221
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3222..4037
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRFAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3295..3314)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(4208..4585,1..211)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
       61 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      121 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      181 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      241 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      301 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      361 gcgcagcgag tcagtgagcg aggaagcgga acagcgccca atacgcaaac cgcctctccc
      421 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      481 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
      541 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      601 aaacagctat gaccatgctc gagccaagct cggcgcgcca ttgggatgat tgactttgct
      661 cgatgctttg tgtgtgataa atgcaaggag ttttaatgta aatgtcatat gtgggagcct
      721 gcacatcatc cccttccaca cacaggcagc atctcctaaa aactcacagg tgggaaaagg
      781 gatgtgcagg tttgtgagac gtggcttcat tgtacatcct tccccatgac cttcatagtt
      841 cccagagcct ttgctggctc ctagtgggct ctcaccacct cactctgcct cctctgttgg
      901 tttgtggtct cctccctgct ggagggtaag tctttgggga gaggagacat tggaaccctt
      961 tcagtcaaag catggggtgg tccctctggc caacttggct agctaccgcc agggaactca
     1021 gtccttggag accacagcca gagggacttg tcattctgag cggtgcccat ctgctgactg
     1081 atatctcacg ggggctcttc tcttcctttg tagagtgcct ggtgaagaat gtgatgggat
     1141 cactagctac gattacaagg atgacgatga caagctaggc tccatggcag aaatcggtac
     1201 tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga
     1261 tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta
     1321 cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct
     1381 gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg
     1441 cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga
     1501 ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat
     1561 tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg
     1621 cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcagaa
     1681 cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat
     1741 ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc
     1801 aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat
     1861 ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct
     1921 gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga
     1981 catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat
     2041 cgcgcgctgg ctgtcgacgc tcgagatttc cggcgagcca accactgagg atctgtactt
     2101 tcagagctct gcagagagcg gccctgggac gagattgaga aatctgccag taatggggga
     2161 tggactagaa acttcccaaa tgtctacaac acaggcccag gcccaacccc agccagccaa
     2221 cgcagccagc accaaccccc cgcccccaga gacctccaac cctaacaagc ccaagaggca
     2281 gaccaaccaa ctgcaatacc tgctcagagt ggtgctcaag acactatgga aacaccagtt
     2341 tgcatggcct ttccagcagc ctgtggatgc cgtcaagctg aacctccctg tgagtagtgt
     2401 gggcagggcc cagggtggcc gtcctggagg tcgtctcctg catgtctcct ggtcagggca
     2461 gttcctgggg ggagggtagg agatgcattt ggaatcatgg tgagagctgc agggcccttg
     2521 agtgagtggt ttcagcagtt ctgatcagtt gaaatcaggg agaaagaatg agctaattgg
     2581 cttaacaata actggtttct tagatatatc ccaaacgcgt caattcactg gccgtcgttt
     2641 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2701 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2761 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     2821 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2881 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2941 catccgctta cagacaagct gtgacagtct ccgggagctg catgtgtcag aggttttcac
     3001 cgtcatcacc gaaacgcgcg agacaaaagg gcctcgtgat acgcctattt ttataggtta
     3061 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3121 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3181 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagccat attcaacggg
     3241 aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     3301 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     3361 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     3421 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     3481 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccctggg aaaacagcat
     3541 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     3601 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     3661 ttcgtttcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     3721 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     3781 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     3841 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     3901 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     3961 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     4021 tcgatgagtt tttctaactg tcagaccaag tttactcata tatactttag attgatttaa
     4081 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4141 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4201 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4261 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4321 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4381 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4441 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4501 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4561 gaacgaccta caccgaactg agata
//
