LOCUS       sequence-386390-        6110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His8-EWS-FLI1(TypeII) 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 6110)
  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 6110)
  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-FLI1_Type2
FEATURES             Location/Qualifiers
     source          1..6110
                     /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(1826..1846)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1853..1876)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             1891..1913
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1944..1963)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1945..1963)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3665..3856
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3850..3872)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3958..4097
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4013..4032
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4112..4129)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4283..4871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4363..4382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5042..5833,5834..5902))
                     /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     5665..5684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5903..6007)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6075..6093
                     /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 gatccgagac ctcaataata gctgcccaga tggctcggca
      361 catgggtatt cggatgacgc ggaacattcg ggttcggata gataccgccg gtcgggctgg
      421 tccaatactg gctcgcggca ccgaaaaagc tgctgctggt caccggcatg ctgctcggat
      481 gcggcggcac aaagttaact ttctgctgat gcgcatgata gctcggcata tagctaatat
      541 cgctcggata tttatacatg ctgctttcgg tcggatgcgg ctgcagggcc tgtgcaatac
      601 catggaaatc gaatttgtac gcgtagcgtt taccatgcac tttggtcatg atgtttttat
      661 cgtagtagta gcgcagggca cggctcagtt tatcgtagtt catgttcggt ttgcttttac
      721 gttcgcccca gcgacgcgca acttcatccg gatcggtcat tttaaattcg ccattggtac
      781 cttcccaggt gatgcagctc gcgttggcgc tatcgctcag cagttccagc agaaactgcc
      841 acagctgaat ctgaccgcta cccggatttg ccaggcggct gctggtcgga cccagaatct
      901 gatacgggtc cggctgcgga cgctgttcgg tgtttttgct aatggtctgc gcaccgccca
      961 gcggcgggct tttattcaga ccgctgttca tattgttgcc ccaggcaccg cgacgaacgc
     1021 tatcatagct cggattctgc tggccataag aagagctctg ctggctatac tggctcggag
     1081 cctggctata gctaccggtc tgcggcggat agctggtcgg cggctgctga ccatagctgc
     1141 tttgttgacc atagctgctc tgctggccat agctgctcgg ctgaccatag gtgttctgct
     1201 ggctatagct gctctgatca tagctggtcg gctgggtgct gctatagctg gtcggcggat
     1261 agctcggcgg tgcggtaacc ggctgcatcg gatagctgcc cggaacctgc ggatagctat
     1321 aattgctctg gccataaccc aggctcggct ggttataacc gccggtgctg ctctgcggct
     1381 ggctggtttc ggtcggtttg ttaccatcct gcgggcgggt cggtgcggtg gccgccggct
     1441 gctgaccata ggccggatat gccggctggg taccataggc gctctgggcc gcatagcttg
     1501 cctgggtggt ggtcacggtg gcggtggtgg tatcataggc accggtacca tagccctgaa
     1561 ccggctggct ataggcctgc ggggcggtcg gggtggtata accggtcggc ggctgaccat
     1621 agctggtggc ataggcggtc tgaccatagg tcgcggtggt ctgggcctgg gtatagctca
     1681 catcggtcgg ctggccatag gtaccatagc tctgctggcc atacgcctgg gtggtctggg
     1741 cataaccctg ggtcggctgc gcggtatagg cgctataacc ctgctgggcc gcggcctggc
     1801 tataggtgct ataatcggtg ctggcgccct gaaaataaag attctcgcta gcatggtgat
     1861 ggtggtggtg atggtggtcc atatggtata tctccttctt aaagttaaac aaaattattt
     1921 ctagagggaa accgttgtgg tctccctata gtgagtcgta ttaatttcgc gggatcgaga
     1981 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgctaca ggtgcggttg
     2041 cttgcgccta tatcgccgac ccggcatcac cggcgctaca ggtgcggttg cttgcgccta
     2101 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
     2161 tttcggcgtg ggtatggtgg caggccccgt ggccgtggga ctgttgggct ccatctcctt
     2221 gcatggggac tgttgggctc catctccttg catgggggga ctgttgggct ccatctcctt
     2281 gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt
     2341 cctaatgcag gagtcgcata agggagagcg tcaaccgatg cccttgagag ccttcaaccc
     2401 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     2461 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     2521 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     2581 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     2641 cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg
     2701 aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc
     2761 gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc
     2821 gctcgcggct cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta
     2881 tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggtgccg ccctatacct
     2941 tgtcatggat tgtaggtgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
     3001 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
     3061 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc
     3121 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
     3181 cagcgttggg tcctggccac gggtgtgcat gatcgtgctc ctgtcgttga ggacccggct
     3241 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     3301 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     3361 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     3421 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     3481 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     3541 accctcacga cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     3601 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     3661 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     3721 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     3781 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     3841 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3901 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3961 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     4021 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     4081 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4141 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4201 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4261 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4321 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4381 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4441 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4501 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4561 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4621 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4681 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4741 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4801 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4861 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4921 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4981 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5041 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5101 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5161 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5221 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5281 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5341 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5401 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5461 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5521 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5581 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5641 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5701 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5761 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5821 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5881 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6001 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     6061 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
//
