LOCUS       sequence-478743-        6220 bp    DNA     circular SYN 16-JUL-2025
DEFINITION  Expresses the RGG1-RRM (300-447) region from the RNA-binding protein
            EWS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6220)
  TITLE     Molecular Basis of EWS Interdomain Self-Association and Its Role in 
            Condensate Formation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6220)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pEmal10H – MBP-EWS_300-447 (RGG1+RRM)
FEATURES             Location/Qualifiers
     source          1..6220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             26..46
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(547..565)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      561..608
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(783..788,789..805,806..811))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(838..856)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        924..1028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1029..1097,1098..1889)
                     /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     complement(1247..1266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2060..2648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2549..2568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2802..2819
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2834..2973
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2899..2918)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3059..3081
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3075..3266)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4968..4987
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4968..4986
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4987..5011
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             5026..5048
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5061..5090
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5097..6197
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     6171..6194
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
ORIGIN
        1 caataacaat aacaacaacc tcggggagaa tctttatttt cagggcgccc gtggtcgtgg
       61 ccgtggtggc tttgatcgtg gtggcatgag ccgtggtggc cgtggtggcg gtcgtggcgg
      121 tatgggtagc gcgggcgagc gtggcggttt caacaaaccg ggcggtccga tggacgaagg
      181 tccggacctg gatctgggtc cgccggttga cccggatgag gacagcgata acagcgcgat
      241 ctacgtgcag ggtctgaacg acagcgttac cctggacgat ctggcggatt tctttaagca
      301 gtgcggcgtg gttaagatga acaaacgtac cggtcaaccg atgatccaca tttacctgga
      361 taaagaaacc ggcaagccga aaggtgacgc gaccgtgagc tacgaggacc cgccgaccgc
      421 gaaggcggcg gttgaatggt tcgacggcaa agattttcag ggtagcaagc tgaaagtgag
      481 cctggcgcgt aagtgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg
      541 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
      601 gttttttgct gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg
      661 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
      721 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
      781 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
      841 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
      901 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      961 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1021 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1081 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1141 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1201 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     1261 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     1321 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     1381 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     1441 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     1501 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     1561 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     1621 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     1681 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     1741 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     1801 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     1861 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1921 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1981 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2041 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2101 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2161 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2221 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2281 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2341 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2401 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2461 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2521 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2581 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2641 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2701 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2761 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2821 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2881 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2941 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     3001 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3061 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3121 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3181 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3241 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3301 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3361 catgcccggt tactggaacg tcgtgagggt aaacaactgg cggtatggat gcggcgggac
     3421 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3481 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3541 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3601 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3661 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3721 atgcacaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     3781 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     3841 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     3901 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     3961 ggtatagggc ggcacctaca atccatgaca aggtataggg cggcacctac aatccatgcc
     4021 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
     4081 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     4141 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     4201 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     4261 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     4321 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4381 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4441 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4501 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggttga
     4561 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     4621 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg agcccaacag tccccccatg
     4681 caaggagatg gagcccaaca gtccccatgc aaggagatgg agcccaacag tcccacggcc
     4741 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     4801 cgatcttccc catcggtgat gtcggcgata taggcgcaag caaccgcacc tgtagcgccg
     4861 gtgatgccgg gtcggcgata taggcgcaag caaccgcacc tgtagcgccg gtgatgccgg
     4921 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     4981 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     5041 gaaggagata tcatatgggc catcatcatc atcatcatca tcatcatcac agcagcatga
     5101 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
     5161 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
     5221 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
     5281 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
     5341 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
     5401 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
     5461 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
     5521 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
     5581 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
     5641 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
     5701 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
     5761 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
     5821 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
     5881 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
     5941 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     6001 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta
     6061 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     6121 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     6181 tgaaagacgc gcagactaat tcgagctcga acaacaacaa
//
