LOCUS       sequence-492503-        7120 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Expresses wild-type H4K16Q-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 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-H4K16Q-Halo-FRT
FEATURES             Location/Qualifiers
     source          1..7120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             419..460
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(498..515)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    504..728
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(861..880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1012..1059
                     /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)."
     CDS             1067..2089
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1067..2089
                     /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
                     "
     polyA_signal    2219..2340
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2256..2275)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2310..2329
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2389..2405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2389..2405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2402..2424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2413..2429
                     /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)."
     promoter        complement(join(2437..2443,2444..2461,2462..2467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2482..2503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2620..2637)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2791..3379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2871..2890)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3550..4341,4342..4410))
                     /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     4173..4192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4411..4515)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4590..4609)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4897..6075
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5128..6066
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6023..6043
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     6092..6111
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6092..6110
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(6607..7120,1..371)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRGGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
ORIGIN
        1 cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc
       61 tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg
      121 agccagcgaa catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg
      181 tcccgaagct gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc
      241 gcctggccaa aagcctgcct aactgcaagg ctgtggacat cggcccgggt ctgaatctgc
      301 tgcaagaaga caacccggac ctgatcggca gcgagatcgc gcgctggctg tctactctgg
      361 agatttccgg ttaagttaat tcggcggccg ctcgagtcta gagggcccgc ggttcgaagg
      421 taagcctatc cctaaccctc tcctcggtct cgattctacg cgtaccggtt agtaatgagt
      481 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
      541 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
      601 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
      661 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
      721 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
      781 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      841 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      901 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      961 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     1021 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     1081 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     1141 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     1201 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     1261 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     1321 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     1381 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     1441 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     1501 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     1561 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     1621 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     1681 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     1741 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     1801 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     1861 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     1921 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     1981 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     2041 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     2101 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     2161 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     2221 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2281 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2341 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     2401 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2461 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2521 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2581 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2641 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2701 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2761 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2821 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2881 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2941 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3001 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3061 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3121 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3181 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3241 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3301 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3361 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3421 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3481 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3541 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3601 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3661 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3721 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3781 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3841 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3901 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3961 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4021 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4081 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4141 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4201 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4261 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4321 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4381 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4441 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4501 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     4561 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4621 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     4681 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     4741 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagg
     4801 cttttgcaaa aagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc
     4861 taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca
     4921 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
     4981 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     5041 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     5101 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc
     5161 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg
     5221 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
     5281 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
     5341 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
     5401 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
     5461 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
     5521 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
     5581 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
     5641 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     5701 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     5761 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     5821 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     5881 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     5941 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     6001 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     6061 tttcaggtgt cgtgaggaat tagcttggta ctaatacgac tcactatagg gagacccaag
     6121 ctggctaggt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
     6181 agatatccag cacagtggcg gccggccgct ctagagctag cgaatttcga gatgtctgga
     6241 cgtggtaagg gcgggaaggg tttgggtaag gggggtgccc agcgccaccg caaggtgttg
     6301 cgtgacaaca tccagggcat caccaagccg gccatccggc gtctggcccg gcgtggcggt
     6361 gtgaagcgga tctctggtct gatctacgag gagactcgcg gggtgctcaa ggtgtttttg
     6421 gagaacgtga tccgtgacgc tgtcacctat acggagcacg ccaagcgcaa gacagtcact
     6481 gccatggacg tggtctacgc gcttaagcgc cagggacgca ccctttatgg ctttggcggt
     6541 gatccatctg gtggaggtgg ttcaggcgga ggtggaagtg gaggtggagg ctctggtggc
     6601 ggttccgaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag
     6661 cgcatgcact acgtcgatgt tggtccgcgc ggtggcaccc ctgtgctgtt cctgcacggt
     6721 aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc
     6781 tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc
     6841 ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc
     6901 gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca
     6961 gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa
     7021 tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag
     7081 ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc
//
