LOCUS       sequence-492439-        8167 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Expresses wild-type H4-Halo under EF1 promoter and can be randomly 
            integrated by PiggyBac transposase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8167)
  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 8167)
  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: PB-EF1-H4-HaloTag-IRES-Neo
FEATURES             Location/Qualifiers
     source          1..8167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(89..193)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(219..674)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(356..377)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     568..587
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     801..823
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     815..832
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     816..832
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   962..1024
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1477..1688
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1701..1969
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2388..3272
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     misc_feature    join(3310..3884,3885..3887,3888..3896)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3477..3494)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3704..3723
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3909..4703
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3963..3982)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4573..4592
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4763..4783)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4825..4946
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4862..4881)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4916..4935
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5709..5743
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6234..6250)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6234..6250)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6247..6269)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6258..6274
                     /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(6282..6288,6289..6306,6307..6312))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6327..6348
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6465..6482)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6636..7224)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6716..6735)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7395..8167,1..19,20..88))
                     /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     8018..8037
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
       61 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
      121 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
      181 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
      241 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
      301 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
      361 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
      421 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
      481 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
      541 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
      601 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
      661 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
      721 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
      781 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc
      841 ctcgttcatt cacgtttttg aacccgtgga ggacgggcag actcgcggtg caaatgtgtt
      901 ttacagcgtg atggagcaga tgaagatgct cgacacgctg cagaacacgc agctagatta
      961 accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgtgt aaaattgacg
     1021 catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt
     1081 ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat
     1141 ttatttatta aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttatg
     1201 agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg
     1261 cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg
     1321 gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac
     1381 gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa aggcctccac
     1441 ggccaaggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     1501 tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga aggtggcgcg
     1561 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     1621 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     1681 gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc cgccctacct
     1741 gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt ggtgcctcct
     1801 gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc ctttgtccgg
     1861 cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg accctgcttg
     1921 ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc aagctgtgac
     1981 cggcgcctac tctagagcta gcgaatttcg agatgtctgg acgtggtaag ggcgggaagg
     2041 gtttgggtaa ggggggtgcc aagcgccacc gcaaggtgtt gcgtgacaac atccagggca
     2101 tcaccaagcc ggccatccgg cgtctggccc ggcgtggcgg tgtgaagcgg atctctggtc
     2161 tgatctacga ggagactcgc ggggtgctca aggtgttttt ggagaacgtg atccgtgacg
     2221 ctgtcaccta tacggagcac gccaagcgca agacagtcac tgccatggac gtggtctacg
     2281 cgcttaagcg ccagggacgc accctttatg gctttggcgg tgatccatct ggtggaggtg
     2341 gttcaggcgg aggtggaagt ggaggtggag gctctggtgg cggttccgaa atcggtactg
     2401 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     2461 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     2521 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     2581 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     2641 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     2701 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     2761 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     2821 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     2881 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     2941 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     3001 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     3061 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     3121 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     3181 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     3241 cgcgctggct gtctactctg gagatttccg gttaagttaa ttcgaattta aatcggatcc
     3301 gcggccgcgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
     3361 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     3421 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     3481 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
     3541 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     3601 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     3661 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     3721 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     3781 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     3841 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgg
     3901 cgtccggaat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3961 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4021 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4081 atgaactgca ggacgaggca gcgcggctat cgtggctggc cgcgacgggc gttccttgcg
     4141 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4201 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4261 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4321 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4381 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4441 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4501 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4561 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4621 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4681 gccttcttga cgagttcttc tgagtcgaca atcaacctct ggattacaaa atttgtgaaa
     4741 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4801 tgcctttgta tcatgcgtta actaaacttg tttattgcag cttataatgg ttacaaataa
     4861 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4921 ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg atgtcaatta
     4981 gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg tttaatattt
     5041 aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac caggttgcag
     5101 ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca ggtttagccc
     5161 cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc cgtatccccc
     5221 caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga tcccgtgcca
     5281 ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc
     5341 ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag ggacgtaatt
     5401 acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga aaatatatat
     5461 ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa
     5521 tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt
     5581 caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac
     5641 tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt
     5701 caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct agctgcatca
     5761 ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc gctatctggg
     5821 catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt
     5881 tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat
     5941 tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg ccacctggga
     6001 taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga agcgcatcag
     6061 caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag
     6121 cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga tgccgcagaa
     6181 atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc atggtcatag
     6241 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     6301 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     6361 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6421 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6481 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6541 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6601 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6661 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6721 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6781 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     6841 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6901 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6961 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7021 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7081 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7141 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7201 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7261 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7321 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7381 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7441 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7501 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7561 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7621 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7681 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7741 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7801 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     7861 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7921 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7981 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     8041 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8101 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8161 tttactt
//
