LOCUS       sequence-492447-        7119 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Expresses wild-type H4(A5,8,12,16)-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 7119)
  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 7119)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 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(A5,8,12,16)-HaloTag-FRT
FEATURES             Location/Qualifiers
     source          1..7119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     853..872
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        947..1051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1052..1120,1121..1912)
                     /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(1270..1289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2083..2671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2572..2591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2825..2842
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2959..2980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2995..3000,3001..3018,3019..3025)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3033..3049
                     /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     3038..3060
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3057..3073
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3057..3073
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3122..3243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3133..3152)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3187..3206
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3373..4395)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(3373..4395)
                     /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(4403..4450)
                     /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     4582..4601
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4734..4958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4947..4964
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5002..5043)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(5094..5978)
                     /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(6470..6489)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6471..6489)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(join(6506..7119,1..565))
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          join(6515..7119,1..334)
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6538..6558)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 ggcccgcatt tacaagacta tcttgccaga aaaaaagcgt cgcagcaggt catcaaaaat
       61 tttaaatggc tagagactta tcgaaagcag cgagacaggc gcgaaggtgc caccagattc
      121 gcacgcggcg gccccagcgc ccaggccagg cctcaactca agcacgaggc gaaggggctc
      181 cttaagcgca aggcctcgaa ctctcccacc cacttccaac ccgaagctcg ggatcaagaa
      241 tcacgtactg cagccaggtg gaagtaattc aaggcacgca agggccataa cccgtaaaga
      301 ggccaggccc gcgggaacca cacacggcac ttacctgtgt tctggcggca aacccgttgc
      361 gaaaaagaac gttcacggcg actactgcac ttatatacgg ttctccccca ccctcgggaa
      421 aaaggcggag ccagtacacg acatcacttt cccagtttac cccgcgccac cttctctagg
      481 caccggttca attgccgacc cctcccccca acttctcggg gactgtgggc gatgtgcgct
      541 ctgcccactg acgggcaccg gagccaattc ccactccttt caagacctag aaggtccatt
      601 agctgcaaag attcctctct gtttaaaact ttatccatct ttgcaaagct ttttgcaaaa
      661 gcctagtcaa taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa
      721 aacgcctaac cctaagcaga ttcttcatgc aattgtcggt caagccttgc cttgttgtag
      781 cttaaatttt gctcgcgcac tactcagcga cctccaacac acaagcaggg agcagatact
      841 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatagg ggatcgggag
      901 atctcccgat ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      961 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1021 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1081 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1141 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1201 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1261 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1321 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1381 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1441 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1501 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1561 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     1621 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1681 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1741 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1801 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1861 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1921 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1981 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2041 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2101 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2161 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2221 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2281 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2341 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2401 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2461 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2521 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2581 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2641 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2701 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2761 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2821 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     2881 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2941 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     3001 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     3061 aaacagctat gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg
     3121 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3181 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     3241 gttggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc
     3301 gtcccggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc
     3361 tcgtagtacg tgctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc
     3421 ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt
     3481 acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc
     3541 tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag
     3601 catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg
     3661 cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta
     3721 ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc
     3781 cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc
     3841 ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc
     3901 catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca
     3961 tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag
     4021 atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc
     4081 ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag
     4141 gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa
     4201 gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac
     4261 atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg
     4321 catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt
     4381 gagttcaggc tttttggcca aggaagttcc tatactttct agagaatagg aacttcggaa
     4441 taggaacttc taggtacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     4501 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     4561 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     4621 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     4681 gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga
     4741 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     4801 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     4861 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     4921 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta
     4981 aactcattac taaccggtac gcgtagaatc gagaccgagg agagggttag ggataggctt
     5041 accttcgaac cgcgggccct ctagactcga gcggcggccg ccgaattaac ttaaccggaa
     5101 atctccagag tagacagcca gcgcgcgatc tcgctgccga tcaggtccgg gttgtcttct
     5161 tgcagcagat tcagacccgg gccgatgtcc acagccttgc agttaggcag gcttttggcc
     5221 aggcgagcgg cttcggccgg tgggatcaga acgcctgggg tgccccagaa cagcagcttc
     5281 gggacagggg actggtgcag ccagtccatg tattcttcga ccagcgcgac gatgttcgct
     5341 ggctcaccgg cgattggcag ctcgtttggg aagcgccaca gtggctcgcg gtcaacagga
     5401 ttcaggaacg gctcgcggta atggtccatc tcgacttcag tcagcgggcg gacgacaccc
     5461 atcggcagcg taccctcgat aaaaacgttc tgatcgatga tcagcttgcg gccgacgtcg
     5521 gtggtgcgga aggcctggaa ggtctcgcgg gcaaattctg gccattcgtc ccaggtcggg
     5581 atagggcgga tgaactccat aaatgcaata cctttgacgc gctctggatt gcgcttggcc
     5641 cagtggaaac ccagagcgga gccccagtcg tgaatgacca ggacgacctc ttccagaccc
     5701 agggcttcga tgaaggcatc catgaagcgg acgtggtcgt cgaagaaata acccaggtct
     5761 ggtttgtcgg atttgcccat accgatcagg tctggagcaa tgcagcgatg ggtcggtgca
     5821 acatgcggga tgatgttgcg ccacacgtag gaggaggtcg ggttaccgtg caggaacagc
     5881 acaggggtgc catcgcgcgg accaacatcg acgtagtgca tgcgctcgcc caggacttcc
     5941 acataatggg ggtcgaatgg aaagccagta ccgatttcgg aaccgccacc agagcctcca
     6001 cctccacttc cacctccgcc tgaaccacct ccaccagatg gatcaccgcc aaagccataa
     6061 agggtgcgtc cctggcgctt aagcgcgtag accacgtcca tggcagtgac tgtcttgcgc
     6121 ttggcgtgct ccgtataggt gacagcgtca cggatcacgt tctccaaaaa caccttgagc
     6181 accccgcgag tctcctcgta gatcagacca gagatccgct tcacaccgcc acgccgggcc
     6241 agacgccgga tggccggctt ggtgatgccc tggatgttgt cacgcaacac cttgcggtgg
     6301 cgcgcggcac cccccgcacc caaacccgcc ccgcccgcac cacgtccaga catctcgaaa
     6361 ttcgctagct ctagagcggc cgccactgtg ctggatatct gcagaattcc accacactgg
     6421 actagtggat ccgagctcgg taccaagctt acctagccag cttgggtctc cctatagtga
     6481 gtcgtattag taccaagcta attcctcacg acacctgaaa tggaagaaaa aaactttgaa
     6541 ccactgtctg aggcttgaga atgaaccaag atccaaactc aaaaagggca aattccaagg
     6601 agaattacat caagtgccaa gctggcctaa cttcagtctc cacccactca gtgtggggaa
     6661 actccatcgc ataaaacccc tccccccaac ctaaagacga cgtactccaa aagctcgaga
     6721 actaatcgag gtgcctggac ggcgcccggt actccgtgga gtcacatgaa gcgacggctg
     6781 aggacggaaa ggcccttttc ctttgtgtgg gtgactcacc cgcccgctct cccgagcgcc
     6841 gcgtcctcca ttttgagctc cctgcagcag ggccgggaag cggccatctt tccgctcacg
     6901 caactggtgc cgaccgggcc agccttgccg cccagggcgg ggcgatacac ggcggcgcga
     6961 ggccaggcac cagagcaggc cggccagctt gagactaccc ccgtccgatt ctcggtggcc
     7021 gcgctcgcag gccccgcctc gccgaacatg tgcgctggga cgcacgggcc ccgtcgccgc
     7081 ccgcggcccc aaaaaccgaa ataccagtgt gcagatctt
//
