LOCUS       sequence-94266-e        6116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Flag-tagged H3.3 (H3F3B) in mammalian cells (CMV promoter). 
            Also suitable for in vitro transcription (T7 promoter) for injecting
            mRNA into oocytes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6116)
  AUTHORS   Inoue A, Zhang Y
  TITLE     Nucleosome assembly is required for nuclear pore complex assembly in
            mouse zygotes.
  JOURNAL   Nat Struct Mol Biol. 2014 Jul;21(7):609-16. doi: 10.1038/nsmb.2839. 
            Epub 2014 Jun 8.
  PUBMED    24908396
REFERENCE   2  (bases 1 to 6116)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nsmb.2839";
            journalName: "Nat Struct Mol Biol"; date: "2014-07"; volume: "21"; 
            issue: "7"; pages: "609-16"
COMMENT     Sequence Label: pcDNA-Flag-H3.3-polyA
FEATURES             Location/Qualifiers
     source          1..6116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             911..934
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1619..1648
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1664..1681
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1704..1721)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1710..1934
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1980..2408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2067..2086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2277..2298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2417..2437)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2422..2752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2603..2738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2665..2684
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2819..3613
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2873..2892)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3483..3502
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3789..3910
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3826..3845)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3880..3899
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3959..3975)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3959..3975)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3972..3994)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3983..3999
                     /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(4007..4013,4014..4031,4032..4037))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4052..4073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4190..4207)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4361..4949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4441..4460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5120..5911,5912..5980))
                     /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     5743..5762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5981..6085)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttatg gactacaaag acgatgacga caagggatcc actagtaacg gccgccagtg
      961 tgctggaatt cacgcccgaa ccaagcagac tgctcgtaag tccaccggtg ggaaagcccc
     1021 ccgcaaacag ctggccacga aagccgccag gaaaagcgct ccctctaccg gcggggtgaa
     1081 gaagcctcat cgctacaggc ccgggaccgt ggcgcttcga gagattcgtc gttatcagaa
     1141 gtcgaccgag ctgctcatcc ggaagctgcc cttccagagg ttggtgaggg agatcgcgca
     1201 ggatttcaaa accgacctga ggtttcagag cgcagccatc ggtgcgctgc aggaggctag
     1261 cgaagcgtac ctggtgggtc tgttcgaaga taccaacctg tgtgccatcc acgctaagag
     1321 agtcaccatc atgcccaaag acatccagtt ggctcgccgg atacggggag agagagctta
     1381 agaattctgc agatatccat cacactggcg gccgctcgac tatgggcacc aaagaacctg
     1441 taaacgttat ctttttaaat tgaatgtgca caaataaaag tttggaaaag aaaaaaaaaa
     1501 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1561 aaaaaaaaaa aaatacgcat catacgctgt ggcgtacctc gagtctagag ggcccttcga
     1621 acaaaaactc atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca
     1681 ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1741 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1801 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1861 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1921 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1981 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2041 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2101 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     2161 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2221 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2281 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2341 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2401 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2461 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2521 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2581 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2641 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     2701 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     2761 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     2821 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2881 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2941 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3001 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3061 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3121 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3181 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3241 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3301 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3361 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3421 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3481 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3541 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3601 cgagttcttc tgagcgggac tctggggttc gcgaaatgac cgaccaagcg acgcccaacc
     3661 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3721 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3781 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3841 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3901 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     3961 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4021 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4081 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4141 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4201 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4261 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4321 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4381 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4441 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4501 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     4561 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4621 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4681 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4741 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4801 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4861 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4921 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4981 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5041 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5101 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5161 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5221 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5281 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5341 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5401 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5461 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5521 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5581 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5641 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5701 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5761 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5821 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5881 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5941 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6001 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6061 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
