LOCUS       sequence-492448-        7118 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Expresses wild-type H4K16R-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 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-H4K16R-Halo-FRT
FEATURES             Location/Qualifiers
     source          1..7118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1116..1135
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1210..1314
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1315..1383,1384..2175)
                     /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(1533..1552)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2346..2934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2835..2854
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3088..3105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3222..3243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3258..3263,3264..3281,3282..3288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3296..3312
                     /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     3301..3323
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3320..3336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3320..3336
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3385..3506
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3396..3415)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3450..3469
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3636..4658)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(3636..4658)
                     /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(4666..4713)
                     /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     4845..4864
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4997..5221
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5210..5227
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5265..5306)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(5354..6238)
                     /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(6732..6751)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6733..6751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(join(6768..7118,1..828))
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          join(6777..7118,1..597)
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6800..6820)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 gctccctgca gcagggccgg gaagcggcca tctttccgct cacgcaactg gtgccgaccg
       61 ggccagcctt gccgcccagg gcggggcgat acacggcggc gcgaggccag gcaccagagc
      121 aggccggcca gcttgagact acccccgtcc gattctcggt ggccgcgctc gcaggccccg
      181 cctcgccgaa catgtgcgct gggacgcacg ggccccgtcg ccgcccgcgg ccccaaaaac
      241 cgaaatacca gtgtgcagat cttggcccgc atttacaaga ctatcttgcc agaaaaaaag
      301 cgtcgcagca ggtcatcaaa aattttaaat ggctagagac ttatcgaaag cagcgagaca
      361 ggcgcgaagg tgccaccaga ttcgcacgcg gcggccccag cgcccaggcc aggcctcaac
      421 tcaagcacga ggcgaagggg ctccttaagc gcaaggcctc gaactctccc acccacttcc
      481 aacccgaagc tcgggatcaa gaatcacgta ctgcagccag gtggaagtaa ttcaaggcac
      541 gcaagggcca taacccgtaa agaggccagg cccgcgggaa ccacacacgg cacttacctg
      601 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
      661 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
      721 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
      781 ggggactgtg ggcgatgtgc gctctgccca ctgacgggca ccggagccaa ttcccactcc
      841 tttcaagacc tagaaggtcc attagctgca aagattcctc tctgtttaaa actttatcca
      901 tctttgcaaa gctttttgca aaagcctagt caataatcaa tgtcaacgcg tatatctggc
      961 ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc
     1021 ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa
     1081 cacacaagca gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     1141 agtgcaccat aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg
     1201 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1261 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1321 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1381 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1441 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1501 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     1561 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1621 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1681 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1741 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1801 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1861 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1921 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1981 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2041 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2101 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2161 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2221 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2281 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2341 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2401 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2461 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     2521 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2581 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2641 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2701 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2761 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2821 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2881 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2941 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3001 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3061 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3121 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     3181 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     3241 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     3301 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta
     3361 gaggtcgacg gtatacagac atgataagat acattgatga gtttggacaa accacaacta
     3421 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3481 ccattataag ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg
     3541 cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag
     3601 aaggcggcgg tggaatcgaa atctcgtagt acgtgctatt cctttgccct cggacgagtg
     3661 ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc
     3721 gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg
     3781 tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag
     3841 agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc tgcaagctcc
     3901 ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag
     3961 aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg
     4021 accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg
     4081 aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg
     4141 acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca
     4201 atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg
     4261 ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatggc ctccgcgacc
     4321 ggctgcagaa cagcgggcag ttcggtttcg ggcaggtctt gcaacgtgac accctgtgca
     4381 cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag cacttccgga
     4441 atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg
     4501 gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga
     4561 gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga
     4621 aacttctcga cagacgtcgc ggtgagttca ggctttttgg ccaaggaagt tcctatactt
     4681 tctagagaat aggaacttcg gaataggaac ttctaggtac gtgaaccatc accctaatca
     4741 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4801 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4861 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4921 gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt
     4981 ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc
     5041 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     5101 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     5161 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     5221 gtcgaggctg atcagcgggt ttaaactcat tactaaccgg tacgcgtaga atcgagaccg
     5281 aggagagggt tagggatagg cttaccttcg aaccgcgggc cctctagact cgagcggccg
     5341 ccgaattaac ttaaccggaa atctccagag tagacagcca gcgcgcgatc tcgctgccga
     5401 tcaggtccgg gttgtcttct tgcagcagat tcagacccgg gccgatgtcc acagccttgc
     5461 agttaggcag gcttttggcc aggcgagcgg cttcggccgg tgggatcaga acgcctgggg
     5521 tgccccagaa cagcagcttc gggacagggg actggtgcag ccagtccatg tattcttcga
     5581 ccagcgcgac gatgttcgct ggctcaccgg cgattggcag ctcgtttggg aagcgccaca
     5641 gtggctcgcg gtcaacagga ttcaggaacg gctcgcggta atggtccatc tcgacttcag
     5701 tcagcgggcg gacgacaccc atcggcagcg taccctcgat aaaaacgttc tgatcgatga
     5761 tcagcttgcg gccgacgtcg gtggtgcgga aggcctggaa ggtctcgcgg gcaaattctg
     5821 gccattcgtc ccaggtcggg atagggcgga tgaactccat aaatgcaata cctttgacgc
     5881 gctctggatt gcgcttggcc cagtggaaac ccagagcgga gccccagtcg tgaatgacca
     5941 ggacgacctc ttccagaccc agggcttcga tgaaggcatc catgaagcgg acgtggtcgt
     6001 cgaagaaata acccaggtct ggtttgtcgg atttgcccat accgatcagg tctggagcaa
     6061 tgcagcgatg ggtcggtgca acatgcggga tgatgttgcg ccacacgtag gaggaggtcg
     6121 ggttaccgtg caggaacagc acaggggtgc catcgcgcgg accaacatcg acgtagtgca
     6181 tgcgctcgcc caggacttcc acataatggg ggtcgaatgg aaagccagta ccgatttcgg
     6241 aaccgccacc agagcctcca cctccacttc cacctccgcc tgaaccacct ccaccagatg
     6301 gatcaccgcc aaagccataa agggtgcgtc cctggcgctt aagcgcgtag accacgtcca
     6361 tggcagtgac tgtcttgcgc ttggcgtgct ccgtataggt gacagcgtca cggatcacgt
     6421 tctccaaaaa caccttgagc accccgcgag tctcctcgta gatcagacca gagatccgct
     6481 tcacaccgcc acgccgggcc agacgccgga tggccggctt ggtgatgccc tggatgttgt
     6541 cacgcaacac cttgcggtgg cgcctggcac cccccttacc caaacccttc ccgcccttac
     6601 cacgtccaga catctcgaaa ttcgctagct ctagagcggc cccgccactg tgctggatat
     6661 ctgcagaatt ccaccacact ggactagtgg atccgagctc ggtaccaagc ttacctagcc
     6721 agcttgggtc tccctatagt gagtcgtatt agtaccaagc taattcctca cgacacctga
     6781 aatggaagaa aaaaactttg aaccactgtc tgaggcttga gaatgaacca agatccaaac
     6841 tcaaaaaggg caaattccaa ggagaattac atcaagtgcc aagctggcct aacttcagtc
     6901 tccacccact cagtgtgggg aaactccatc gcataaaacc cctcccccca acctaaagac
     6961 gacgtactcc aaaagctcga gaactaatcg aggtgcctgg acggcgcccg gtactccgtg
     7021 gagtcacatg aagcgacggc tgaggacgga aaggcccttt tcctttgtgt gggtgactca
     7081 cccgcccgct ctcccgagcg ccgcgtcctc cattttga
//
