LOCUS       sequence-376803-        4874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His8-FLI1_DBD_deltaAlpha4(276-361) 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 4874)
  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 4874)
  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_deltaAlpha4
FEATURES             Location/Qualifiers
     source          1..4874
                     /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(590..610)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(617..640)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             655..677
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(708..727)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(709..727)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2429..2620
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2614..2636)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2722..2861
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2777..2796
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2876..2893)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3047..3635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3127..3146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3806..4597,4598..4666))
                     /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     4429..4448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4667..4771)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4839..4857
                     /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 gatcctcaat cgaatttgta cgcgtagcgt ttaccatgca
      361 ctttggtcat gatgttttta tcgtagtagt agcgcagggc acggctcagt ttatcgtagt
      421 tcatgttcgg tttgctttta cgttcgcccc agcgacgcgc aacttcatcc ggatcggtca
      481 ttttaaattc gccattggta ccttcccagg tgatgcagct cgcgttggcg ctatcgctca
      541 gcagttccag cagaaactgc cacagctgaa tctgaccgct acccggggcg ccctgaaaat
      601 aaagattctc gctagcatgg tgatggtggt ggtgatggtg gtccatatgg tatatctcct
      661 tcttaaagtt aaacaaaatt atttctagag ggaaaccgtt gtggtctccc tatagtgagt
      721 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
      781 tcaccggcgc tacaggtgcg gttgcttgcg cctatatcgc cgacccggca tcaccggcgc
      841 tacaggtgcg gttgcttgcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
      901 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgt
      961 gggactgttg ggctccatct ccttgcatgg ggactgttgg gctccatctc cttgcatggg
     1021 gggactgttg ggctccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     1081 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcaacc
     1141 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     1201 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     1261 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     1321 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     1381 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta
     1441 cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc
     1501 ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga
     1561 ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcattgg
     1621 accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg
     1681 gattgtaggt gccgccctat accttgtcat ggattgtagg tgccgcccta taccttgtct
     1741 gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg
     1801 gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag
     1861 aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag
     1921 cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgt gcatgatcgt
     1981 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     2041 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     2101 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     2161 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     2221 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     2281 ccgcatccat accgccagtt gtttaccctc acgacgttcc agtaaccggg catgttcatc
     2341 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     2401 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     2461 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     2521 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     2581 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     2641 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     2701 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     2761 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg
     2821 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     2881 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2941 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3001 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3061 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3121 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3181 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3241 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3301 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3361 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3421 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3481 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3541 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3601 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3661 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3721 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3781 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3841 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3901 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3961 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4021 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4081 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     4141 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4201 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4261 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4321 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4381 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     4441 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4501 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4561 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4621 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4681 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4741 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4801 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4861 ttcgtcttca agaa
//
