LOCUS       sequence-252751-        7538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of histone H3.3K4M-FLAG/HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7538)
  AUTHORS   Jang Y, Broun A, Wang C, Park YK, Zhuang L, Lee JE, Froimchuk E, Liu
            C, Ge K
  TITLE     H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and 
            impairs adipose tissue development.
  JOURNAL   Nucleic Acids Res. 2019 Jan 25;47(2):607-620. doi: 
            10.1093/nar/gky982.
  PUBMED    30335158
REFERENCE   2  (bases 1 to 7538)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2019-01-25- 25"; volume: "47"; issue: 
            "2"; pages: "607-620"
COMMENT     Sequence Label: pBT346.6-H3.3-K4M-bGHpolyA
FEATURES             Location/Qualifiers
     source          1..7538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..583
                     /label=chicken beta-actin promoter
     intron          584..1600
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1524..1546
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1608..1627
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1686..1719
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1721..1739)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1883..2017
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2137..2271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     intron          2367..2432
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2562..2582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3007..3128
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3171..3204
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3694..3717
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3733..3759
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             3733..3759
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(3825..3843)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3826..3843)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3863..3880)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3869..4093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4122..4246
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4265..4283)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4304..4320)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4304..4320)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4317..4339)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4328..4344
                     /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(4352..4358,4359..4376,4377..4382))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4397..4418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4535..4552)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4706..5294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4786..4805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5465..6256,6257..6325))
                     /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     6088..6107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6326..6430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6456..6911)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6593..6614)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6805..6824
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7038..7060
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7052..7069
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7053..7069
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7076..7094
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7139..7238
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    7266..7335
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      361 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      421 gggggggcgc gcgccrggsg gggsggggsg gggsgrgggg sggggsgggg cgaggcggag
      481 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
      541 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
      601 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
      661 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg
      721 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg
      781 ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg
      841 gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg
      901 gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg
      961 cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     1021 agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc
     1081 tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt
     1141 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg
     1201 ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
     1261 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     1321 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
     1381 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
     1441 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
     1501 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     1561 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     1621 tattgtgctg tctcatcatt ttggcaaaga attcgcgcgt cttaattaac gactcactat
     1681 agggcataac ttcgtataat gtatgctata cgaagttatc cctttagtga gggttaattg
     1741 cagatctgcg actctagagg atctgcgact ctagaggatc ataatcagcc ataccacatt
     1801 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     1861 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     1921 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     1981 gtccaaactc atcaatgtat cttatcatgt ctggatctgc gactctagag gatcataatc
     2041 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     2101 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     2161 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2221 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccggccctgt
     2281 ctggtcgagg gatctttgtg aaggaacctt acttctgtgg tgtgacataa ttggacaaac
     2341 tacctacaga gatttaaagc tctaaggtaa atataaaatt tttaagtgta taatgtgtta
     2401 aactactgat tctaattgtt tgtgtatttt agattccaac ctatggaact gatgaatggg
     2461 agcagtggtg gaatgccttt aatgaggaaa acctgttttg ctcagaagaa atgccatcta
     2521 gtgatgatga ggctactgct gactctcaac attctactcc tccaaaaaag aagagaaagg
     2581 tagaagaccc caaggacttt ccttcagaat tgctaagttt tttgagtcat gctgtgttta
     2641 gtaatagaac tcttgcttgc tttgctattt acaccacaaa ggaaaaagct gcactgctat
     2701 acaagaaaat tatggaaaaa tattctgtaa cctttataag taggcataac agttataatc
     2761 ataacatact gttttttctt actccacaca ggcatagagt gtctgctatt aataactatg
     2821 ctcaaaaatt gtgtaccttt agctttttaa tttgtaaagg ggttaataag gaatatttga
     2881 tgtatagtgc cttgactaga gatcataatc agccatacca catttgtaga ggttttactt
     2941 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3001 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3061 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3121 gtatcttatc acccggggga tccccatcaa gctgatccgg aacccttaat ataacttcgt
     3181 ataatgtatg ctatacgaag ttattaggtc cctcgacctg ctgcagctcg aggcatgcgt
     3241 cgacgtttct agtctagact cgagtgaacc atggcccgaa ccatgcagac tgctcgtaag
     3301 tccaccggtg ggaaagcccc ccgcaaacag ctggccacga aagccgccag gaaaagcgct
     3361 ccctctaccg gcggggtgaa gaagcctcat cgctacaggc ccgggaccgt ggcgcttcga
     3421 gagattcgtc gttatcagaa gtcgaccgag ctgctcatcc ggaagctgcc cttccagagg
     3481 ttggtgaggg agatcgcgca ggatttcaaa accgacctga ggtttcagag cgcagccatc
     3541 ggtgcgctgc aggaggctag cgaagcgtac ctggtgggtc tgttcgaaga taccaacctg
     3601 tgtgccatcc acgctaagag agtcaccatc atgcccaaag acatccagtt ggctcgccgg
     3661 atacggggag agagagctgc ggccgctgga ggagactaca aggacgacga tgacaagtcg
     3721 gccgctggag gataccccta cgacgtgccc gactacgcct aggaattcgt taacgaaatc
     3781 catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata gtgtcaccta
     3841 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3901 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3961 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     4021 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     4081 gtgggctcta tggcgcgccc aactagttct agagcggccg cacaagtttg tacaaaaaag
     4141 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     4201 aacagactac ataatactgt aaaacacaac atatccagtc actatggagc tccagctttt
     4261 gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg
     4321 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4381 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4441 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4501 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4561 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4621 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4681 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4741 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4801 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4861 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4921 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4981 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5041 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5101 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5161 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5221 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5281 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5341 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5401 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5461 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5521 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5581 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5641 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5701 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5761 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5821 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5881 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5941 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6001 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6061 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6121 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6181 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6241 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6301 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6361 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6421 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     6481 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6541 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6601 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6661 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6721 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6781 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6841 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6901 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     6961 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     7021 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata
     7081 cgactcacta tagggcgaat tgggtaccag ttacgctagg gataacaggg taatatagca
     7141 aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg
     7201 cttttttata atgccaactt tgtacaaaaa agcaggctct cgaggtcgac gatgtaggtc
     7261 acggtctcga agccgcggtg cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac
     7321 tccacctcac ccatctggtc catcatgatg aacgggtcga ggtggcggta gttgatcccg
     7381 gcgaacgcgc ggcgcaccgg gaagccctcg ccctcgaaac cgctgggcgc ggtggtcacg
     7441 gtgagcacgg gacgtgcgac ggcgtcggcg ggtgcggata cgcggggcag cgtcagcggg
     7501 ttctcgacgg tcacggcggg cmyrtmkayg gwgttccg
//
