LOCUS       sequence-376754-        4925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His8-PU.1_DBD(168-270) 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 4925)
  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 4925)
  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-PU.1_DBD(168-270)
FEATURES             Location/Qualifiers
     source          1..4925
                     /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(641..661)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(668..691)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             706..728
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(759..778)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(760..778)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2480..2671
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2665..2687)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2773..2912
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2828..2847
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2927..2944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3098..3686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3178..3197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3857..4648,4649..4717))
                     /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     4480..4499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4718..4822)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4890..4908
                     /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 gatccttagt gcggcgggtg acgacgttcc gccaggccac
      361 cacgacccag aacctcaccg ctaaactgat aggtcagctt tttcttaact ttcttcactt
      421 cgccggtttt accatagtta cgcagcgcac gcgccatctt ttggtaggtc attttcttac
      481 ggttgccttt ctggataccc caacggtgcg ccagcgcctc tttgtgtttg ctgctaaatt
      541 ggaaggtgcc cttatctttg tccacccacc aaatgctatc cttcatgtca ccgctacgca
      601 gcaggtccag caggaactgg tacagacgga ttttcttggc gccctgaaaa taaagattct
      661 cgctagcatg gtgatggtgg tggtgatggt ggtccatatg gtatatctcc ttcttaaagt
      721 taaacaaaat tatttctaga gggaaaccgt tgtggtctcc ctatagtgag tcgtattaat
      781 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
      841 ctacaggtgc ggttgcttgc gcctatatcg ccgacccggc atcaccggcg ctacaggtgc
      901 ggttgcttgc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
      961 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg tgggactgtt
     1021 gggctccatc tccttgcatg gggactgttg ggctccatct ccttgcatgg ggggactgtt
     1081 gggctccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     1141 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcaac cgatgccctt
     1201 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     1261 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     1321 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     1381 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     1441 cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct
     1501 ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg
     1561 catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg
     1621 acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat
     1681 cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg
     1741 tgccgcccta taccttgtca tggattgtag gtgccgccct ataccttgtc tgcctccccg
     1801 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     1861 cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa
     1921 tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac
     1981 gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg tgcatgatcg tgctcctgtc
     2041 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     2101 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     2161 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     2221 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     2281 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     2341 taccgccagt tgtttaccct cacgacgttc cagtaaccgg gcatgttcat catcagtaac
     2401 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     2461 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     2521 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     2581 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     2641 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     2701 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2761 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     2821 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     2881 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     2941 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3001 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3061 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3121 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3181 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3241 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3301 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3361 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3421 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3481 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     3541 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3601 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3661 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3721 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3781 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3841 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3901 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3961 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4021 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4081 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4141 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     4201 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4261 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4321 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4381 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4441 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     4501 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4561 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4621 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4681 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4741 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4801 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4861 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     4921 aagaa
//
