LOCUS       sequence-488105-        5080 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag mouse histone H3.1 with Halo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5080)
  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 5080)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 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-T_mouseH3.1-HaloTag
FEATURES             Location/Qualifiers
     source          1..5080
                     /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             2969..3853
                     /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(4643..4661)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4644..4661)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4679..4695)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4679..4695)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4692..4714)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4703..4719
                     /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(4727..4733,4734..4751,4752..4757))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4772..4793
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4910..4927)
                     /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 aattgctcgt actaagcaga ccgctcgcaa
     2521 gtccaccggt ggcaaggccc cgcgtaagca gctggctacc aaggccgccc gcaagagcgc
     2581 cccggccacc ggcggcgtga agaagcctca ccgctaccgt cccggcaccg tggcgctgcg
     2641 cgagatccgg cgctaccaga agtcgaccga gctgctgatc cgcaagctgc cgttccagcg
     2701 cctggtgcgc gagatcgcgc aggacttcaa gaccgacctg cgcttccaga gctcggccgt
     2761 catggctctg caggaggcct gtgaggccta ccttgtgggt ctgtttgagg acaccaacct
     2821 gtgcgccatc cacgccaagc gtgtcaccat catgcccaag gacatccaac tggcccgccg
     2881 catccgcggg gagagggcgg gaggttctgg cggaagtggt ggaggtggat ctggtggagg
     2941 tggatctggt ggcggcggtt caggatccga aatcggtact ggctttccat tcgaccccca
     3001 ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc gcgatggcac
     3061 ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca acatcatccc
     3121 gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg gcaaatccga
     3181 caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg ccttcatcga
     3241 agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg ctctgggttt
     3301 ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg agttcatccg
     3361 ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc aggccttccg
     3421 caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacgct
     3481 gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc gcgagccgtt
     3541 cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg
     3601 tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc accagtcccc
     3661 tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg ccgaagccgc
     3721 tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg gtctgaatct
     3781 gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc tgtctactct
     3841 ggagatttcc ggttaataat tgtacagttc cgtacagttc tacggaatta accaaaggct
     3901 cttttcagag ccacacacga tttcaagtaa aatggctgta atttactcca tccttaaacg
     3961 aacctatatg ttccctagtg tctataacgg tggatattcc ccaaaacttg agtttataac
     4021 agcccaaagt tgatatcaag tacaaaatag gtcaactgag tgtctggttt tacccgtgga
     4081 atatgcgtaa agtgtacggg tcggatctct gacgtgacct gctcgcaggc tttcggtcct
     4141 taaagggctc cgatccagag aggggtctgt gatgtcactt ttcccgccgt ccggctttcg
     4201 gggcggggct gatgggcgag atatgtgaca tcactttccc gccggcgcgc tttctggtcg
     4261 tgactgaggg gcggaatttg tgaagtcact ttcccgccgg cgggcttttg cgtctttccc
     4321 ggcatggctg agaggaggga tcactgatgt cgctttcccg ccggcgcgct ttcgggtctt
     4381 ggctgagggg cggggtttgt gatgtcattt tcccgccggc gcgctttcgg ggcatggttg
     4441 atgggaggga tttgtgacgt cactttcccg cccgggcgct ttcggttctt tcccgccagg
     4501 tcttattggc ggggtttatg acgtcaccgt ccggcgggcg ggttttcgtt tgtgtgattc
     4561 tgttgggggt gggagatcaa ggccaagcag ctatcgacca tatgggagag ctcccaacgc
     4621 gttggatgca tagcttgagt attctatagt gtcacctaaa tagcttggcg taatcatggt
     4681 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4741 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4801 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4861 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4921 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4981 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5041 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
//
