LOCUS       sequence-373725-        5405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His8-EWS 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 5405)
  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 5405)
  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-EWS
FEATURES             Location/Qualifiers
     source          1..5405
                     /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(1121..1141)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1148..1171)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             1186..1208
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1239..1258)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1240..1258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2960..3151
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3145..3167)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3253..3392
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3308..3327
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3407..3424)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3578..4166)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3658..3677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4337..5128,5129..5197))
                     /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     4960..4979
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5198..5302)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5370..5388
                     /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 gatcctcact gctggccata agaagagctc tgctggctat
      361 actggctcgg agcctggcta tagctaccgg tctgcggcgg atagctggtc ggcggctgct
      421 gaccatagct gctttgttga ccatagctgc tctgctggcc atagctgctc ggctgaccat
      481 aggtgttctg ctggctatag ctgctctgat catagctggt cggctgggtg ctgctatagc
      541 tggtcggcgg atagctcggc ggtgcggtaa ccggctgcat cggatagctg cccggaacct
      601 gcggatagct ataattgctc tggccataac ccaggctcgg ctggttataa ccgccggtgc
      661 tgctctgcgg ctggctggtt tcggtcggtt tgttaccatc ctgcgggcgg gtcggtgcgg
      721 tggccgccgg ctgctgacca taggccggat atgccggctg ggtaccatag gcgctctggg
      781 ccgcatagct tgcctgggtg gtggtcacgg tggcggtggt ggtatcatag gcaccggtac
      841 catagccctg aaccggctgg ctataggcct gcggggcggt cggggtggta taaccggtcg
      901 gcggctgacc atagctggtg gcataggcgg tctgaccata ggtcgcggtg gtctgggcct
      961 gggtatagct cacatcggtc ggctggccat aggtaccata gctctgctgg ccatacgcct
     1021 gggtggtctg ggcataaccc tgggtcggct gcgcggtata ggcgctataa ccctgctggg
     1081 ccgcggcctg gctataggtg ctataatcgg tgctcgcggc gccctgaaaa taaagattct
     1141 cgctagcatg gtgatggtgg tggtgatggt ggtccatatg gtatatctcc ttcttaaagt
     1201 taaacaaaat tatttctaga gggaaaccgt tgtggtctcc ctatagtgag tcgtattaat
     1261 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     1321 ctacaggtgc ggttgcttgc gcctatatcg ccgacccggc atcaccggcg ctacaggtgc
     1381 ggttgcttgc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     1441 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg tgggactgtt
     1501 gggctccatc tccttgcatg gggactgttg ggctccatct ccttgcatgg ggggactgtt
     1561 gggctccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     1621 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcaac cgatgccctt
     1681 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     1741 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
     1801 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
     1861 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
     1921 cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct
     1981 ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg
     2041 catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg
     2101 acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat
     2161 cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg
     2221 tgccgcccta taccttgtca tggattgtag gtgccgccct ataccttgtc tgcctccccg
     2281 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     2341 cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa
     2401 tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac
     2461 gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg tgcatgatcg tgctcctgtc
     2521 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     2581 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     2641 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     2701 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     2761 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     2821 taccgccagt tgtttaccct cacgacgttc cagtaaccgg gcatgttcat catcagtaac
     2881 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     2941 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     3001 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     3061 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     3121 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3181 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3241 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     3301 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     3361 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     3421 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3481 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3541 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3601 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3661 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3721 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3781 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3841 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3901 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3961 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagra
     4021 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4081 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4141 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4201 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4261 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4321 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4381 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4441 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4501 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4561 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4621 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     4681 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4741 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4801 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4861 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4921 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     4981 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5041 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5101 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5161 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5221 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5281 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5341 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     5401 aagaa
//
