LOCUS       sequence-491700-        7117 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Expresses wild-type H4K16A-Halo under EF1 promoter and can be 
            integrated into FRT site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7117)
  AUTHORS   Semeigazin A, Iida S, Minami K, Tamura S, Ide S, Higashi K, Toyoda 
            A, Kurokawa K, Maeshima K
  TITLE     Behaviors of nucleosomes with mutant histone H4s in euchromatic 
            domains of living human cells.
  JOURNAL   Histochem Cell Biol. 2024 Jul;162(1-2):23-40. doi: 
            10.1007/s00418-024-02293-x. Epub 2024 May 14.
  PUBMED    38743310
REFERENCE   2  (bases 1 to 7117)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Histochem 
            Cell Biol."; date: "2024-07"; volume: "162(1-2):23-40. doi"; pages: 
            " 10.1007/s00418-024-02293-x. Epub 2024 May 14"
COMMENT     Sequence Label: pEF5-H4K16A-Halo-FRT
FEATURES             Location/Qualifiers
     source          1..7117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..28
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    145..166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(181..186,187..204,205..211)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    219..235
                     /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     224..246
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     243..259
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     243..259
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    308..429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(319..338)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     373..392
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(559..1581)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(559..1581)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     protein_bind    complement(1589..1636)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     1768..1787
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1920..2144
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2133..2150
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2188..2229)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(2277..3161)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(3655..3674)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3656..3674)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3691..4868)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3700..4637
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3723..3743)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5156..5175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5250..5354
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5355..5423,5424..6215)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     LVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5573..5592)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6386..6974
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6875..6894
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
       61 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      121 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      181 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca
      301 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
      361 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
      421 aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca
      481 gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg cggtggaatc
      541 gaaatctcgt agtacgtgct attcctttgc cctcggacga gtgctggggc gtcggtttcc
      601 actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat
      661 ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc
      721 ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat
      781 gcggagcata tacgcccgga gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa
      841 gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac
      901 ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc
      961 attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg
     1021 gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt
     1081 gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc
     1141 acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg
     1201 gctaagatcg gccgcagcga tcgcatccat ggcctccgcg accggctgca gaacagcggg
     1261 cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata
     1321 ggtcaggctc tcgctgaatt ccccaatgtc aagcacttcc ggaatcggga gcgcggccga
     1381 tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg
     1441 caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga
     1501 gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt
     1561 cgcggtgagt tcaggctttt tggccaagga agttcctata ctttctagag aataggaact
     1621 tcggaatagg aacttctagg tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     1681 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     1741 aacgccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     1801 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     1861 gctacagggc gcgtggggat accccctaga gccccagctg gttctttccg cctcagaagc
     1921 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct
     1981 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat
     2041 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac
     2101 gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg
     2161 ggtttaaact cattactaac cggtacgcgt agaatcgaga ccgaggagag ggttagggat
     2221 aggcttacct tcgaaccgcg ggccctctag actcgagcgg ccgccgaatt aacttaaccg
     2281 gaaatctcca gagtagacag ccagcgcgcg atctcgctgc cgatcaggtc cgggttgtct
     2341 tcttgcagca gattcagacc cgggccgatg tccacagcct tgcagttagg caggcttttg
     2401 gccaggcgag cggcttcggc cggtgggatc agaacgcctg gggtgcccca gaacagcagc
     2461 ttcgggacag gggactggtg cagccagtcc atgtattctt cgaccagcgc gacgatgttc
     2521 gctggctcac cggcgattgg cagctcgttt gggaagcgcc acagtggctc gcggtcaaca
     2581 ggattcagga acggctcgcg gtaatggtcc atctcgactt cagtcagcgg gcggacgaca
     2641 cccatcggca gcgtaccctc gataaaaacg ttctgatcga tgatcagctt gcggccgacg
     2701 tcggtggtgc ggaaggcctg gaaggtctcg cgggcaaatt ctggccattc gtcccaggtc
     2761 gggatagggc ggatgaactc cataaatgca atacctttga cgcgctctgg attgcgcttg
     2821 gcccagtgga aacccagagc ggagccccag tcgtgaatga ccaggacgac ctcttccaga
     2881 cccagggctt cgatgaaggc atccatgaag cggacgtggt cgtcgaagaa ataacccagg
     2941 tctggtttgt cggatttgcc cataccgatc aggtctggag caatgcagcg atgggtcggt
     3001 gcaacatgcg ggatgatgtt gcgccacacg taggaggagg tcgggttacc gtgcaggaac
     3061 agcacagggg tgccatcgcg cggaccaaca tcgacgtagt gcatgcgctc gcccaggact
     3121 tccacataat gggggtcgaa tggaaagcca gtaccgattt cggaaccgcc accagagcct
     3181 ccacctccac ttccacctcc gcctgaacca cctccaccag atggatcacc gccaaagcca
     3241 taaagggtgc gtccctggcg cttaagcgcg tagaccacgt ccatggcagt gactgtcttg
     3301 cgcttggcgt gctccgtata ggtgacagcg tcacggatca cgttctccaa aaacaccttg
     3361 agcaccccgc gagtctcctc gtagatcaga ccagagatcc gcttcacacc gccacgccgg
     3421 gccagacgcc ggatggccgg cttggtgatg ccctggatgt tgtcacgcaa caccttgcgg
     3481 tggcgcgcgg cacccccctt acccaaaccc ttcccgccct taccacgtcc agacatctcg
     3541 aaattcgcta gctctagagc ggccccgcca ctgtgctgga tatctgcaga attccaccac
     3601 actggactag tggatccgag ctcggtacca agcttaccta gccagcttgg gtctccctat
     3661 agtgagtcgt attagtacca agctaattcc tcacgacacc tgaaatggaa gaaaaaaact
     3721 ttgaaccact gtctgaggct tgagaatgaa ccaagatcca aactcaaaaa gggcaaattc
     3781 caaggagaat tacatcaagt gccaagctgg cctaacttca gtctccaccc actcagtgtg
     3841 gggaaactcc atcgcataaa acccctcccc ccaacctaaa gacgacgtac tccaaaagct
     3901 cgagaactaa tcgaggtgcc tggacggcgc ccggtactcc gtggagtcac atgaagcgac
     3961 ggctgaggac ggaaaggccc ttttcctttg tgtgggtgac tcacccgccc gctctcccga
     4021 gcgccgcgtc ctccattttg agctccctgc agcagggccg ggaagcggcc atctttccgc
     4081 tcacgcaact ggtgccgacc gggccagcct tgccgcccag ggcggggcga tacacggcgg
     4141 cgcgaggcca ggcaccagag caggccggcc agcttgagac tacccccgtc cgattctcgg
     4201 tggccgcgct cgcaggcccc gcctcgccga acatgtgcgc tgggacgcac gggccccgtc
     4261 gccgcccgcg gcccaaaaac cgaaatacca gtgtgcagat cttggcccgc atttacaaga
     4321 ctatcttgcc agaaaaaaag cgtcgcagca ggtcatcaaa aattttaaat ggctagagac
     4381 ttatcgaaag cagcgagaca ggcgcgaagg tgccaccaga ttcgcacgcg gcggccccag
     4441 cgcccaggcc aggcctcaac tcaagcacga ggcgaagggg ctccttaagc gcaaggcctc
     4501 gaactctccc acccacttcc aacccgaagc tcgggatcaa gaatcacgta ctgcagccag
     4561 gtggaagtaa ttcaaggcac gcaagggcca taacccgtaa agaggccagg cccgcgggaa
     4621 ccacacacgg cacttacctg tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg
     4681 gcgactactg cacttatata cggttctccc ccaccctcgg gaaaaaggcg gagccagtac
     4741 acgacatcac tttcccagtt taccccgcgc caccttctct aggcaccggt tcaattgccg
     4801 acccctcccc ccaacttctc ggggactgtg ggcgatgtgc gctctgccca ctgacgggca
     4861 ccggagccaa ttcccactcc tttcaagacc tagaaggtcc attagctgca aagattcctc
     4921 tctgtttaaa actttatcca tctttgcaaa gctttttgca aaagcctagt caataatcaa
     4981 tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc
     5041 agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg
     5101 cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa ctatgcggca
     5161 tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc gatccgtcga
     5221 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5281 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5341 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5401 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5461 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5521 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5581 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5641 ctcagaatga cttggttgag tactcactag tcacagaaaa gcatcttacg gatggcatga
     5701 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5761 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5821 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5881 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5941 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6001 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6061 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6121 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6181 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6241 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6301 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6361 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6421 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6481 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6541 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6601 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6661 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6721 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6781 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6841 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6901 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6961 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7021 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7081 cctttgagtg agctgatacc gctcgccgca gccgaac
//
