LOCUS       sequence-491706-        7118 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  Expresses wild-type H4-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 7118)
  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 7118)
  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-HaloTag-FRT
FEATURES             Location/Qualifiers
     source          1..7118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     346..365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     599..616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    733..754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(769..774,775..792,793..799)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    807..823
                     /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     812..834
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     831..847
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     831..847
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    896..1017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(907..926)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     961..980
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1147..2169)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(1147..2169)
                     /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(2177..2224)
                     /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     2356..2375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2508..2732
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2721..2738
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2776..2817)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(2865..3749)
                     /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(4243..4262)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4244..4262)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4279..5457)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4288..5226
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4311..4331)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5745..5764
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5839..5943
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5944..6012,6013..6804)
                     /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(6162..6181)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6975..7118,1..445)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
       61 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
      121 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
      181 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
      241 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
      301 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
      361 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
      421 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      481 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
      541 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
      601 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
      661 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      721 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      781 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      841 ctatgaccat gattacgcca agctctagct agaggtcgac ggtatacaga catgataaga
      901 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
      961 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttggg
     1021 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg
     1081 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtag
     1141 tacgtgctat tcctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag
     1201 tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc
     1261 gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc
     1321 atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata
     1381 cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg
     1441 ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga
     1501 atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag
     1561 gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc
     1621 ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac
     1681 agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt
     1741 gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc
     1801 cgcagcgatc gcatccatgg cctccgcgac cggctgcaga acagcgggca gttcggtttc
     1861 aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc
     1921 gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg
     1981 ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc
     2041 acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag
     2101 gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc
     2161 aggctttttg gccaaggaag ttcctatact ttctagagaa taggaacttc ggaataggaa
     2221 cttctaggta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2281 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2341 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2401 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2461 gtggggatac cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac
     2521 cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac
     2581 cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta
     2641 ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca
     2701 aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaactca
     2761 ttactaaccg gtacgcgtag aatcgagacc gaggagaggg ttagggatag gcttaccttc
     2821 gaaccgcggg ccctctagac tcgagcggcc gccgaattaa cttaaccgga aatctccaga
     2881 gtagacagcc agcgcgcgat ctcgctgccg atcaggtccg ggttgtcttc ttgcagcaga
     2941 ttcagacccg ggccgatgtc cacagccttg cagttaggca ggcttttggc caggcgagcg
     3001 gcttcggccg gtgggatcag aacgcctggg gtgccccaga acagcagctt cgggacaggg
     3061 gactggtgca gccagtccat gtattcttcg accagcgcga cgatgttcgc tggctcaccg
     3121 gcgattggca gctcgtttgg gaagcgccac agtggctcgc ggtcaacagg attcaggaac
     3181 ggctcgcggt aatggtccat ctcgacttca gtcagcgggc ggacgacacc catcggcagc
     3241 gtaccctcga taaaaacgtt ctgatcgatg atcagcttgc ggccgacgtc ggtggtgcgg
     3301 aaggcctgga aggtctcgcg ggcaaattct ggccattcgt cccaggtcgg gatagggcgg
     3361 atgaactcca taaatgcaat acctttgacg cgctctggat tgcgcttggc ccagtggaaa
     3421 cccagagcgg agccccagtc gtgaatgacc aggacgacct cttccagacc cagggcttcg
     3481 atgaaggcat ccatgaagcg gacgtggtcg tcgaagaaat aacccaggtc tggtttgtcg
     3541 gatttgccca taccgatcag gtctggagca atgcagcgat gggtcggtgc aacatgcggg
     3601 atgatgttgc gccacacgta ggaggaggtc gggttaccgt gcaggaacag cacaggggtg
     3661 ccatcgcgcg gaccaacatc gacgtagtgc atgcgctcgc ccaggacttc cacataatgg
     3721 gggtcgaatg gaaagccagt accgatttcg gaaccgccac cagagcctcc acctccactt
     3781 ccacctccgc ctgaaccacc tccaccagat ggatcaccgc caaagccata aagggtgcgt
     3841 ccctggcgct taagcgcgta gaccacgtcc atggcagtga ctgtcttgcg cttggcgtgc
     3901 tccgtatagg tgacagcgtc acggatcacg ttctccaaaa acaccttgag caccccgcga
     3961 gtctcctcgt agatcagacc agagatccgc ttcacaccgc cacgccgggc cagacgccgg
     4021 atggccggct tggtgatgcc ctggatgttg tcacgcaaca ccttgcggtg gcgcttggca
     4081 ccccccttac ccaaaccctt cccgccctta ccacgtccag acatctcgaa attcgctagc
     4141 tctagagcgg ccccgccact gtgctggata tctgcagaat tccaccacac tggactagtg
     4201 gatccgagct cggtaccaag cttacctagc cagcttgggt ctccctatag tgagtcgtat
     4261 tagtaccaag ctaattcctc acgacacctg aaatggaaga aaaaaacttt gaaccactgt
     4321 ctgaggcttg agaatgaacc aagatccaaa ctcaaaaagg gcaaattcca aggagaatta
     4381 catcaagtgc caagctggcc taacttcagt ctccacccac tcagtgtggg gaaactccat
     4441 cgcataaaac ccctcccccc aacctaaaga cgacgtactc caaaagctcg agaactaatc
     4501 gaggtgcctg gacggcgccc ggtactccgt ggagtcacat gaagcgacgg ctgaggacgg
     4561 aaaggccctt ttcctttgtg tgggtgactc acccgcccgc tctcccgagc gccgcgtcct
     4621 ccattttgag ctccctgcag cagggccggg aagcggccat ctttccgctc acgcaactgg
     4681 tgccgaccgg gccagccttg ccgcccaggg cggggcgata cacggcggcg cgaggccagg
     4741 caccagagca ggccggccag cttgagacta cccccgtccg attctcggtg gccgcgctcg
     4801 caggccccgc ctcgccgaac atgtgcgctg ggacgcacgg gccccgtcgc cgcccgcggc
     4861 cccaaaaacc gaaataccag tgtgcagatc ttggcccgca tttacaagac tatcttgcca
     4921 gaaaaaaagc gtcgcagcag gtcatcaaaa attttaaatg gctagagact tatcgaaagc
     4981 agcgagacag gcgcgaaggt gccaccagat tcgcacgcgg cggccccagc gcccaggcca
     5041 ggcctcaact caagcacgag gcgaaggggc tccttaagcg caaggcctcg aactctccca
     5101 cccacttcca acccgaagct cgggatcaag aatcacgtac tgcagccagg tggaagtaat
     5161 tcaaggcacg caagggccat aacccgtaaa gaggccaggc ccgcgggaac cacacacggc
     5221 acttacctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg cgactactgc
     5281 acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca cgacatcact
     5341 ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga cccctccccc
     5401 caacttctcg gggactgtgg gcgatgtgcg ctctgcccac tgacgggcac cggagccaat
     5461 tcccactcct ttcaagacct agaaggtcca ttagctgcaa agattcctct ctgtttaaaa
     5521 ctttatccat ctttgcaaag ctttttgcaa aagcctagtc aataatcaat gtcaacgcgt
     5581 atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat
     5641 gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc
     5701 gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat
     5761 tgtactgaga gtgcaccata ggggatcggg agatctcccg atccgtcgac gtcaggtggc
     5821 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5881 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5941 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6001 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6061 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6121 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6181 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6241 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6301 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6361 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6421 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6481 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6541 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6601 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6661 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6721 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6781 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6841 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6901 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6961 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7021 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7081 aaggtaactg gcttcagcag agcgcagata ccaaatac
//
