LOCUS       sequence-341218-        7749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RNA-binding mutant EZH2 expression plasmid with 3XFLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7749)
  AUTHORS   Long Y, Hwang T, Gooding AR, Goodrich KJ, Rinn JL, Cech TR
  TITLE     RNA is essential for PRC2 chromatin occupancy and function in human 
            pluripotent stem cells.
  JOURNAL   Nat Genet. 2020 Sep;52(9):931-938. doi: 10.1038/s41588-020-0662-x. 
            Epub 2020 Jul 6.
  PUBMED    32632336
REFERENCE   2  (bases 1 to 7749)
  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/s41588-020-0662-x"; journalName: "Nat Genet"; date: 
            "2020-09"; volume: "52"; issue: "9"; pages: "931-938"
COMMENT     Sequence Label: pcDNA3.1_3xFlagEzh2 RNA binding mutant
FEATURES             Location/Qualifiers
     source          1..7749
                     /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             910..975
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3340..3357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3346..3570
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3616..4044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3703..3722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3913..3934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4053..4073)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4058..4387
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4238..4373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4300..4319
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4454..5248
                     /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(4508..4527)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5118..5137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5422..5543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5459..5478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5513..5532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5592..5608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5592..5608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5605..5627)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5616..5632
                     /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(5640..5646,5647..5664,5665..5670))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5685..5706
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5823..5840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5994..6582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6074..6093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6753..7544,7545..7613))
                     /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     7376..7395
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7614..7718)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gctggctagc
      901 gagatgggtg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag
      961 gatgacgatg acaagggtac cggccagact gggaagaaat ctgagaaggg accagtttgt
     1021 tggcggaagc gtgtaaaatc agagtacatg cgactgagac agctcaagag ggccagagct
     1081 gctgctgaag tagccagtat gtttagttcc aatcgtcaga aaattttgga aagaacggaa
     1141 atcttaaacc aagaatggaa acagcgaagg atacagcctg tgcacatcct gacttctgtg
     1201 agctcattgc gcgggactag ggagtgttcg gtgaccagtg acttggattt tccaacacaa
     1261 gtcatcccat taaagactct gaatgcagtt gcttcagtac ccataatgta ttcttggtca
     1321 cccctacagc agaattttat ggtggaagat gaaactgttt tacataacat tccttatatg
     1381 ggagatgaag ttttagatca ggatggtact ttcattgaag aactaataaa aaattatgat
     1441 gggaaagtac acggggatag agaatgtggg tttataaatg atgaaatttt tgtggagttg
     1501 gtgaatgccc ttggtcaata taatgatgat gacgatgatg atgatggaga cgatcctgaa
     1561 gaaagagaag aaaagcagaa agatctggag gatcaccgag atgataaaga aagccgccca
     1621 cctcggaaat ttccttctga taaaattttt gaagccattt cctcaatgtt tccagataag
     1681 ggcacagcag aagaactaaa ggaaaaatat aaagaactca ccgaacagca gctcccaggc
     1741 gcacttcctc ctgaatgtac ccccaacata gatggaccaa atgctaaatc tgttcagaga
     1801 gagcaaagct tacactcctt tcatacgctt ttctgtaggc gatgttttaa atatgactgc
     1861 ttcctacatc gtaagtgcaa ttattctttt catgcaacac ccaacactta taagcggaag
     1921 aacacagaaa cagctctaga caacaaacct tgtggaccac agtgttacca gcatttggag
     1981 ggagcaaagg agtttgctgc tgctctcacc gctgagcgga taaagacccc accaaaacgt
     2041 ccaggaggcc gcagaagagg acggcttccc aataacagta gcaggcccag cacccccacc
     2101 attaatgtgc tggaatcaaa ggatacagac agtgataggg aagcagggac tgaaacgggg
     2161 ggagagaaca atgataaaga agaagaagag aagaaagatg aaacttcgag ctcctctgaa
     2221 gcaaattctc ggtgtcaaac accaataaag atgaagccaa atattgaacc tcctgagaat
     2281 gtggagtgga gtggtgctga agcctcaatg tttagagtcc tcattggcac ttactatgac
     2341 aatttctgtg ccattgctag gttaattggg accaaaacat gtagacaggt gtatgagttt
     2401 agagtcaaag aatctagcat catagctcca gctcccgctg aggatgtgga tactcctaac
     2461 gccgctatcc ggagcaaaca ccggttgtgg gctgcacact gcagaaagat acagctgaaa
     2521 aaggacggct cctctaacca tgtttacaac tatcaaccct gtgatcatcc acggcagcct
     2581 tgtgacagtt cgtgcccttg tgtgatagca caaaattttt gtgaaaagtt ttgtcaatgt
     2641 agttcagagt gtcaaaaccg ctttccggga tgccgctgca aagcacagtg caacaccaag
     2701 cagtgcccgt gctacctggc tgtccgagag tgtgaccctg acctctgtct tacttgtgga
     2761 gccgctgacc attgggacag taaaaatgtg tcctgcaaga actgcagtat tcagcggggc
     2821 tccaaaaagc atctattgct ggcaccatct gacgtggcag gctgggggat ttttatcaaa
     2881 gatcctgtgc agaaaaatga attcatctca gaatactgtg gagagattat ttctcaagat
     2941 gaagctgaca gaagagggaa agtgtatgat aaatacatgt gcagctttct gttcaacttg
     3001 aacaatgatt ttgtggtgga tgcaacccgc aagggtaaca aaattcgttt tgcaaatcat
     3061 tcggtaaatc caaactgcta tgcaaaagtt atgatggtta acggtgatca caggataggt
     3121 atttttgcca agagagccat ccagactggc gaagagctgt tttttgatta cagatacagc
     3181 caggctgatg ccctgaagta tgtcggcatc gaaagagaaa tggaaatccc ttgacgtacc
     3241 gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggc
     3301 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3361 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3421 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3481 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3541 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3601 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3661 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3721 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3781 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3841 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3901 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3961 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4021 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     4081 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4141 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4201 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4261 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4321 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4381 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     4441 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4501 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4561 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4621 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4681 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4741 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4801 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4861 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4921 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4981 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     5041 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     5101 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5161 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5221 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5281 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5341 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5401 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5461 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5521 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5581 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5641 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5701 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5761 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5821 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5881 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5941 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6001 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6061 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6121 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6181 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6241 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6301 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6361 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6421 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6481 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6541 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6601 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6661 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6721 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6781 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6841 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6901 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6961 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7021 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7081 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7141 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7201 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7261 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7321 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7381 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7441 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7501 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7561 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7621 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7681 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7741 cctgacgtc
//
