LOCUS       sequence-491705-        7120 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Expresses wild-type H4(R5,8,12,16)-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 7120)
  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 7120)
  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-H4(R5,8,12,16)-HaloTag-FRT
FEATURES             Location/Qualifiers
     source          1..7120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(55..1077)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(55..1077)
                     /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(1085..1132)
                     /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     1264..1283
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1416..1640
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1629..1646
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1684..1725)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1775..2659)
                     /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(3153..3172)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3154..3172)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3189..4367)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3198..4136
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3221..3241)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     4655..4674
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4749..4853
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4854..4922,4923..5714)
                     /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(5072..5091)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5885..6473
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6374..6393
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6627..6644
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6761..6782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6797..6802,6803..6820,6821..6827)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6835..6851
                     /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     6840..6862
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6859..6875
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6859..6875
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6924..7045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6935..6954)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6989..7008
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtagta cgtgctattc
       61 ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta cttctacaca
      121 gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc
      181 tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc
      241 gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg cccggagccg
      301 cggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct gctccataca
      361 agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat ccccgaacat
      421 cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga cattgttgga
      481 gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc aaagcatcag
      541 ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag tttgccagtg
      601 atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt attgaccgat
      661 tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg cagcgatcgc
      721 atccatggcc tccgcgaccg gctgcagaac agcgggcagt tcggtttcag gcaggtcttg
      781 caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc tgaattcccc
      841 aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat aaacataacg
      901 atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac gccctcctac
      961 atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt cggagacgct
     1021 gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag gctttttggc
     1081 caaggaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tctaggtacg
     1141 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     1201 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     1261 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     1321 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc
     1381 cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
     1441 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     1501 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     1561 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     1621 ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaactcatt actaaccggt
     1681 acgcgtagaa tcgagaccga ggagagggtt agggataggc ttaccttcga accgcgggcc
     1741 ctctagactc gagcggggcc gccgaattaa cttaaccgga aatctccaga gtagacagcc
     1801 agcgcgcgat ctcgctgccg atcaggtccg ggttgtcttc ttgcagcaga ttcagacccg
     1861 ggccgatgtc cacagccttg cagttaggca ggcttttggc caggcgagcg gcttcggccg
     1921 gtgggatcag aacgcctggg gtgccccaga acagcagctt cgggacaggg gactggtgca
     1981 gccagtccat gtattcttcg accagcgcga cgatgttcgc tggctcaccg gcgattggca
     2041 gctcgtttgg gaagcgccac agtggctcgc ggtcaacagg attcaggaac ggctcgcggt
     2101 aatggtccat ctcgacttca gtcagcgggc ggacgacacc catcggcagc gtaccctcga
     2161 taaaaacgtt ctgatcgatg atcagcttgc ggccgacgtc ggtggtgcgg aaggcctgga
     2221 aggtctcgcg ggcaaattct ggccattcgt cccaggtcgg gatagggcgg atgaactcca
     2281 taaatgcaat acctttgacg cgctctggat tgcgcttggc ccagtggaaa cccagagcgg
     2341 agccccagtc gtgaatgacc aggacgacct cttccagacc cagggcttcg atgaaggcat
     2401 ccatgaagcg gacgtggtcg tcgaagaaat aacccaggtc tggtttgtcg gatttgccca
     2461 taccgatcag gtctggagca atgcagcgat gggtcggtgc aacatgcggg atgatgttgc
     2521 gccacacgta ggaggaggtc gggttaccgt gcaggaacag cacaggggtg ccatcgcgcg
     2581 gaccaacatc gacgtagtgc atgcgctcgc ccaggacttc cacataatgg gggtcgaatg
     2641 gaaagccagt accgatttcg gaaccgccac cagagcctcc acctccactt ccacctccgc
     2701 ctgaaccacc tccaccagat ggatcaccgc caaagccata aagggtgcgt ccctggcgct
     2761 taagcgcgta gaccacgtcc atggcagtga ctgtcttgcg cttggcgtgc tccgtatagg
     2821 tgacagcgtc acggatcacg ttctccaaaa acaccttgag caccccgcga gtctcctcgt
     2881 agatcagacc agagatccgc ttcacaccgc cacgccgggc cagacgccgg atggccggct
     2941 tggtgatgcc ctggatgttg tcacgcaaca ccttgcggtg gcgcctggca ccccccctac
     3001 ccaaacccct cccgccccta ccacgtccag acatctcgaa attcgctagc tctagagcgg
     3061 ccccgccact gtgctggata tctgcagaat tccaccacac tggactagtg gatccgagct
     3121 cggtaccaag cttacctagc cagcttgggt ctccctatag tgagtcgtat tagtaccaag
     3181 ctaattcctc acgacacctg aaatggaaga aaaaaacttt gaaccactgt ctgaggcttg
     3241 agaatgaacc aagatccaaa ctcaaaaagg gcaaattcca aggagaatta catcaagtgc
     3301 caagctggcc taacttcagt ctccacccac tcagtgtggg gaaactccat cgcataaaac
     3361 ccctcccccc aacctaaaga cgacgtactc caaaagctcg agaactaatc gaggtgcctg
     3421 gacggcgccc ggtactccgt ggagtcacat gaagcgacgg ctgaggacgg aaaggccctt
     3481 ttcctttgtg tgggtgactc acccgcccgc tctcccgagc gccgcgtcct ccattttgag
     3541 ctccctgcag cagggccggg aagcggccat ctttccgctc acgcaactgg tgccgaccgg
     3601 gccagccttg ccgcccaggg cggggcgata cacggcggcg cgaggccagg caccagagca
     3661 ggccggccag cttgagacta cccccgtccg attctcggtg gccgcgctcg caggccccgc
     3721 ctcgccgaac atgtgcgctg ggacgcacgg gccccgtcgc cgcccgcggc cccaaaaacc
     3781 gaaataccag tgtgcagatc ttggcccgca tttacaagac tatcttgcca gaaaaaaagc
     3841 gtcgcagcag gtcatcaaaa attttaaatg gctagagact tatcgaaagc agcgagacag
     3901 gcgcgaaggt gccaccagat tcgcacgcgg cggccccagc gcccaggcca ggcctcaact
     3961 caagcacgag gcgaaggggc tccttaagcg caaggcctcg aactctccca cccacttcca
     4021 acccgaagct cgggatcaag aatcacgtac tgcagccagg tggaagtaat tcaaggcacg
     4081 caagggccat aacccgtaaa gaggccaggc ccgcgggaac cacacacggc acttacctgt
     4141 gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac
     4201 ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt
     4261 accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg
     4321 gggactgtgg gcgatgtgcg ctctgcccac tgacgggcac cggagccaat tcccactcct
     4381 ttcaagacct agaaggtcca ttagctgcaa agattcctct ctgtttaaaa ctttatccat
     4441 ctttgcaaag ctttttgcaa aagcctagtc aataatcaat gtcaacgcgt atatctggcc
     4501 cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat gcaattgtcg
     4561 gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc gacctccaac
     4621 acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4681 gtgcaccata ggggatcggg agatctcccg atccgtcgac gtcaggtggc acttttcggg
     4741 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4801 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4861 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4921 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4981 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5041 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5101 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5161 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5221 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5281 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5341 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5401 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5461 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5521 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5581 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5641 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5701 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5761 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5821 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5881 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5941 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6001 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6061 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6121 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6181 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6241 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6301 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6361 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6421 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6481 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6541 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6601 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6661 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     6721 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     6781 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     6841 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag
     6901 aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     6961 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     7021 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     7081 gctggaggat catccagccg gcgtcccgga aaacgattcc
//
