LOCUS       sequence-376801-        4988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His8-FLI1_DBD_R2L2(276-399_R337L_R340L_F362A) 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 4988)
  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 4988)
  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-FLI1_DBD_R2L2
FEATURES             Location/Qualifiers
     source          1..4988
                     /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(704..724)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(731..754)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             769..791
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(822..841)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(823..841)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2543..2734
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2728..2750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2836..2975
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2891..2910
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2990..3007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3161..3749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3241..3260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3920..4711,4712..4780))
                     /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     4543..4562
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4781..4885)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4953..4971
                     /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 gatcctcagt taactttctg ctgatgcgca tgatagctcg
      361 gcatatagct aatatcgctc ggatatttat acatgctgct ttcggtcgga tgcggctgca
      421 gggcctgtgc aataccatgg gcatcgaatt tgtacgcgta gcgtttacca tgcactttgg
      481 tcatgatgtt tttatcgtag tagtagagca gggcaaggct cagtttatcg tagttcatgt
      541 tcggtttgct tttacgttcg ccccagcgac gcgcaacttc atccggatcg gtcattttaa
      601 attcgccatt ggtaccttcc caggtgatgc agctcgcgtt ggcgctatcg ctcagcagtt
      661 ccagcagaaa ctgccacagc tgaatctgac cgctacccgg ggcgccctga aaataaagat
      721 tctcgctagc atggtgatgg tggtggtgat ggtggtccat atggtatatc tccttcttaa
      781 agttaaacaa aattatttct agagggaaac cgttgtggtc tccctatagt gagtcgtatt
      841 aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
      901 gcgctacagg tgcggttgct tgcgcctata tcgccgaccc ggcatcaccg gcgctacagg
      961 tgcggttgct tgcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
     1021 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgtgggact
     1081 gttgggctcc atctccttgc atggggactg ttgggctcca tctccttgca tggggggact
     1141 gttgggctcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
     1201 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc aaccgatgcc
     1261 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     1321 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
     1381 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     1441 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     1501 tttcggcgag aagcaggcca ttatcgccgg catggcggcc gacgcgctgg gctacgtctt
     1561 gctggcgttc gcgacgcgag gctggatggc cttccccatt atgattcttc tcgcttccgg
     1621 cggcatcggg atgcccgcgt tgcaggccat gctgtccagg caggtagatg acgaccatca
     1681 gggacagctt caaggatcgc tcgcggctct taccagccta acttcgatca ttggaccgct
     1741 gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg aacgggttgg catggattgt
     1801 aggtgccgcc ctataccttg tcatggattg taggtgccgc cctatacctt gtctgcctcc
     1861 ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca
     1921 cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt
     1981 gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg
     2041 cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgtgcatga tcgtgctcct
     2101 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     2161 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     2221 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     2281 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     2341 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     2401 ccataccgcc agttgtttac cctcacgacg ttccagtaac cgggcatgtt catcatcagt
     2461 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     2521 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     2581 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     2641 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     2701 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2761 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2821 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     2881 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     2941 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     3001 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3061 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3121 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3181 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3241 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3301 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3361 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3421 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3481 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3541 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3601 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3661 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3721 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3781 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3841 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3901 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3961 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4021 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4081 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4141 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4201 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     4261 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4321 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4381 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4441 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4501 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     4561 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4621 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4681 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4741 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4801 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4861 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     4921 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     4981 ttcaagaa
//
