LOCUS       sequence-373719-        4991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His8-ERG_DBD(306-429_F392A) with a TEV site for removal of
            the His8 tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4991)
  AUTHORS   Selig EE, Sohn EJ, Stoja A, Moreno-Romero AK, Akula S, Xu X, Bishop 
            AJR, Libich DS
  TITLE     Phase separation of the oncogenic fusion protein EWS::FLI1 is 
            modulated by its DNA-binding domain.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 May 20;122(20):e2221823122. doi: 
            10.1073/pnas.2221823122. Epub 2025 May 16.
  PUBMED    40377985
REFERENCE   2  (bases 1 to 4991)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2025-05-20"; volume: "122(20):e2221823122. 
            doi"; pages: " 10.1073/pnas.2221823122. Epub 2025 May 16"
COMMENT     Sequence Label: pAG8Ha-ERG_DBD(306-429_F392A)
FEATURES             Location/Qualifiers
     source          1..4991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..33,34..39)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(214..261)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     257..275
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(707..727)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(734..757)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             772..794
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(825..844)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(826..844)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2546..2737
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2731..2753)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2839..2978
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2894..2913
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2993..3010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3164..3752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3244..3263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3923..4714,4715..4783))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4546..4565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4784..4888)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4956..4974
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatccgata agctttaatg cggtagttta tcacagttaa
       61 attgctaacg cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg
      121 gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg ccgggcctct
      181 tgcgggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga
      241 ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt
      301 tcgggctttg ttagcagccg gatccttatt agttcatttt ttgcgggtgc gcgtggtagc
      361 tgcccatata cggcagatcg ctcgggtact tatacaggct gctttccggc gggtgcggtt
      421 gcagcgcttg cgcaatacca tgcgcatcaa atttgtacgc ataacgttta ccgtgaacct
      481 tggtcatgat gttcttatcg tagtagtaac gcagcgcacg gctcagcttg tcatagttca
      541 tgttcggttt gctcttgcgt tcaccccaac gacgcgccac ctcatccggg tcggtcatct
      601 taaattcgcc gttggtaccc tcccaggtaa tgcagctgct gttgctgctg tcgctcagca
      661 gctccagcag gaactgccac agttggatct ggccgctacc cggggcgccc tgaaaataaa
      721 gattctcgct agcatggtga tggtggtggt gatggtggtc catatggtat atctccttct
      781 taaagttaaa caaaattatt tctagaggga aaccgttgtg gtctccctat agtgagtcgt
      841 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
      901 ccggcgctac aggtgcggtt gcttgcgcct atatcgccga cccggcatca ccggcgctac
      961 aggtgcggtt gcttgcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     1021 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccgtggg
     1081 actgttgggc tccatctcct tgcatgggga ctgttgggct ccatctcctt gcatgggggg
     1141 actgttgggc tccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct
     1201 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcaaccgat
     1261 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     1321 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     1381 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     1441 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     1501 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt
     1561 cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc
     1621 cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca
     1681 tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcattggacc
     1741 gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat
     1801 tgtaggtgcc gccctatacc ttgtcatgga ttgtaggtgc cgccctatac cttgtctgcc
     1861 tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg
     1921 gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac
     1981 tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag
     2041 ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgtgca tgatcgtgct
     2101 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     2161 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     2221 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     2281 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     2341 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     2401 catccatacc gccagttgtt taccctcacg acgttccagt aaccgggcat gttcatcatc
     2461 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     2521 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     2581 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     2641 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg
     2701 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     2761 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     2821 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     2881 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg cggtgtgaaa
     2941 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     3001 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3061 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3601 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3781 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3841 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3901 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4021 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4081 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4201 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     4261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     4561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4741 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4861 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     4921 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     4981 gtcttcaaga a
//
