LOCUS       sequence-488011-        5022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag human histone H3.2 with Halo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5022)
  AUTHORS   Minami K, Nakazato K, Ide S, Kaizu K, Higashi K, Tamura S, Toyoda A,
            Takahashi K, Kurokawa K, Maeshima K
  TITLE     Replication-dependent histone labeling dissects the physical 
            properties of euchromatin/heterochromatin in living human cells.
  JOURNAL   Sci Adv. 2025 Mar 28;11(13):eadu8400. doi: 10.1126/sciadv.adu8400. 
            Epub 2025 Mar 28.
  PUBMED    40153514
REFERENCE   2  (bases 1 to 5022)
  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"; journalName: "Sci Adv."; 
            date: "2025-03-28"; volume: "11(13):eadu8400. doi"; pages: " 
            10.1126/sciadv.adu8400. Epub 2025 Mar 28"
COMMENT     Sequence Label: pGEM_H3.2-HaloTag
FEATURES             Location/Qualifiers
     source          1..5022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1803..2258)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1940..1961)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2152..2171
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2384..2406
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2398..2415
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2399..2415
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2422..2441
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2422..2440
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2471..2480
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2475..2484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2952..3836
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     promoter        complement(4585..4603)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4586..4603)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4621..4637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4621..4637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4634..4656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4645..4661
                     /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(4669..4675,4676..4693,4694..4699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4714..4735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4852..4869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     1801 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     1861 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     1921 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     1981 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2041 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2101 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2161 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2221 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc
     2281 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     2341 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     2401 aaaacgacgg ccagtgaatt gtaatacgac tcactatagg gcgaattggg cccgacgtcg
     2461 catgctcccg gccgccatgg cggccgcggg aattcgcccg tactaagcag actgctcgca
     2521 agtcgaccgg cggcaaggcc ccgaggaagc agctggccac caaggcggcc cgcaagagcg
     2581 cgccggccac gggcggggtg aagaagccgc accgctaccg gcccggcacc gtagccctgc
     2641 gggagatccg gcgctaccag aagtccacgg agctgctgat ccgcaagctg cccttccagc
     2701 ggctggtacg cgagatcgcg caggacttta agacggacct gcgcttccag agctcggccg
     2761 tgatggcgct gcaggaggcc agcgaggcct acctggtggg gctgttcgaa gacacgaacc
     2821 tgtgcgccat ccacgccaag cgcgtgacca ttatgcccaa ggacatccag ctggcccgcc
     2881 gcatccgtgg agagcgagct ggtggaggtg gatctggtgg aggtggatct ggtggcggcg
     2941 gttcaggatc cgaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg
     3001 gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc
     3061 acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc
     3121 atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt
     3181 atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag
     3241 aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca
     3301 atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg
     3361 acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc
     3421 gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc
     3481 gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc
     3541 gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg
     3601 tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt
     3661 tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc
     3721 tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc
     3781 cggacctgat cggcagcgag atcgcgcgct ggctgtctac tctggagatt tccggttaat
     3841 aagaagtggc ggttcggccg gaggttccat cgtatccaaa aggctctttt cagagccacc
     3901 cacatcagca cttggaagaa gctgtaccgc ttgccctccg tgctcctccg gcattagagc
     3961 ggggaaggca cttccgctta ggctcccaac aaaacggggt cgggtccccc tgctgcggtg
     4021 ccgtcttgag ctttgccgac ttcggccaat tggcctatcc gcactggccg ggctgcggac
     4081 gctttcctgg gcgccggctc gcgggctttg gcggtccggc tccccccgcg cgtgaccggg
     4141 gccctgcgtg gcgggggagc cggggcagga gagacggggc gtcgggcacg aggccagggg
     4201 tcactagggg gacgctcagg gctcgcggtc ctgagcgcgg ctggcctttg cggaagtgga
     4261 gcgccacacg cccgtgcttc gcgcccaaac cggcctcacc cggcgcggcc atccggctag
     4321 ggagggaaat gaccacctcg gcctcgccgg ggttctgcgg gcaaacgtgg ggagaactgc
     4381 gctggggctg ccgttcagtc cccgcgccac ctccactagc acagtggtaa cccccatatc
     4441 ctgcccctaa gccaaaattc ccgggggcac ggggtgtggg cggaattttg tgaatctggg
     4501 ctttatcgag ccgttccctg tccatgtcca cagagtcgac catatgggag agctcccaac
     4561 gcgttggatg catagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg
     4621 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4681 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4741 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4801 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4861 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tt
//
