LOCUS       sequence-488600-        5382 bp    DNA     circular SYN 07-OCT-2025
DEFINITION  CRISPR/Cas9 donor plasmid to tag human CDC45 with Halo tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5382)
  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 5382)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 7, 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_CDC45-HaloTag
FEATURES             Location/Qualifiers
     source          1..5382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    130..151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(166..171,172..189,190..196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    204..220
                     /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     209..231
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     228..244
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     228..244
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        262..280
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     262..279
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(1078..1962)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     regulatory      2741..2750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2745..2754
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2784..2803)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2785..2803)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2810..2827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2810..2826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2819..2841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2967..3422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3054..3073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3264..3285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3500..3604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3605..3673,3674..4465)
                     /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     complement(3823..3842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4636..5224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5125..5144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5378..5382,1..13)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
       61 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      121 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      181 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      241 tgaccatgat tacgccaagc tatttaggtg acactataga atactcaagc tatgcatcca
      301 acgcgttggg agctctccca tatggtcgac tgcaacagag tcttggcagg gcccatctgc
      361 cacagccgca gcctccaggc tagggctctg ggagatgcag ggctggatgt ggcattccct
      421 gtggggaacc tgggctgaga gatggttaca aagacacaag gcaggcctat ttcctgggaa
      481 cccttaagaa agaggtagca catggggcag tgggaggtgt cgcgggacag tgacctgcca
      541 gtgaatccca atggggccct caggacagct gtgtgctggt gaaggcccca gcagaaccag
      601 cgctttggaa ttcccagcta agcggctgcc aacagctttg ctttctcatg agccaagcac
      661 acaaaagccc ggatggctgc tctgcgggct cttgcccagc gcctgacacg aaggctgtgg
      721 gacaggaggc tccaggtggc cttcgagggc agcaccaggg agtctgtgtg tgacatgtgt
      781 cacacgcgat gcccagccca agtgtggtca gcagagggtg aataccagct ctttctgaag
      841 ggatcgcagg ggcttaatga aattagctgc ctcaaaatgc cccattgtgg ccctgctggc
      901 agcctaagga cagatgtgct tgggaaaaat gtgttggctc caatctcaag aaccctgctt
      961 cggtctgaaa cccaagtaga agcctccgtt gagggaggac agagcagggc atcagggtcg
     1021 ggctctgagg gggctgctgc agccacacct gcagaacccc tgtaactcac cctattaacc
     1081 ggaaatctcc agagtagaca gccagcgcgc gatctcgctg ccgatcaggt ccgggttgtc
     1141 ttcttgcagc agattcagac ccgggccgat gtccacagcc ttgcagttag gcaggctttt
     1201 ggccaggcga gcggcttcgg ccggtgggat cagaacgcct ggggtgcccc agaacagcag
     1261 cttcgggaca ggggactggt gcagccagtc catgtattct tcgaccagcg cgacgatgtt
     1321 cgctggctca ccggcgattg gcagctcgtt tgggaagcgc cacagtggct cgcggtcaac
     1381 aggattcagg aacggctcgc ggtaatggtc catctcgact tcagtcagcg ggcggacgac
     1441 acccatcggc agcgtaccct cgataaaaac gttctgatcg atgatcagct tgcggccgac
     1501 gtcggtggtg cggaaggcct ggaaggtctc gcgggcaaat tctggccatt cgtcccaggt
     1561 cgggataggg cggatgaact ccataaatgc aatacctttg acgcgctctg gattgcgctt
     1621 ggcccagtgg aaacccagag cggagcccca gtcgtgaatg accaggacga cctcttccag
     1681 acccagggct tcgatgaagg catccatgaa gcggacgtgg tcgtcgaaga aataacccag
     1741 gtctggtttg tcggatttgc ccataccgat caggtctgga gcaatgcagc gatgggtcgg
     1801 tgcaacatgc gggatgatgt tgcgccacac gtaggaggag gtcgggttac cgtgcaggaa
     1861 cagcacaggg gtgccatcgc gcggaccaac atcgacgtag tgcatgcgct cgcccaggac
     1921 ttccacataa tgggggtcga atggaaagcc agtaccgatt tcggatcctg atccgccgcc
     1981 accgcttccg ccacctccag agccacctcc acctgacagg agggaaataa gtgcgtccag
     2041 aaacttgctc cgatcctcag ctttcagctc aattactgaa gtaatgaaag aaaaacagcc
     2101 gtgagaaggg aggagtggga acacaagaca gggcacagaa ctcccctccg ctgcctgccc
     2161 tccactgcat tccaaagtgc gcggctcctc ccggcctgtg gtattctgca ctcagtggtt
     2221 gtgtctgtct cactgtactt catcttcaca actgctgtct gcactgctag agaggtcagg
     2281 gttggggcct gcagggagaa ctcccccccg ggaagacaat agaggtgaca cagggaaggg
     2341 ccctacagac ctggacaaac acgggcctgc ccaccccagc tggcatgaac ccccgtcccc
     2401 agagtgggtt gtggaagtcc ttgggtgggg caggtcactt ttggccgcac tctggccaat
     2461 tcctcggctt cagtgttctc ttccgcacaa ggggacatgg tgcaaactgc ccatatctca
     2521 cagggccctc aggctcagct gagcacccag accctcacca caaagcaaaa ccaggcccca
     2581 ggatggggct ggcctggccc tgggagccat ctatgctgcc agccacagcc ccgagagccc
     2641 cagacccagc ccacgcaggt gggcactaga catggcccac tctgcaccct cacccctggc
     2701 tctccaccct cagggaaacc aagcagttcc aattcccgcg gccgccatgg cggccgggag
     2761 catgcgacgt cgggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg
     2821 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2881 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2941 agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3001 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3061 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3121 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3181 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3241 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3301 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3361 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3421 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catcaggtgg
     3481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3601 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3661 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3721 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3781 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3841 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3901 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3961 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4021 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4081 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4141 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4201 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4261 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4321 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4381 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4441 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4501 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4741 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5221 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5281 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5341 agctgatacc gctcgccgca gccgaacgac cgagcgcagc ga
//
