LOCUS       sequence-478744-        6046 bp    DNA     circular SYN 16-JUL-2025
DEFINITION  Expresses the RGG3 (549-638) region from the RNA-binding protein 
            EWS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6046)
  TITLE     Molecular Basis of EWS Interdomain Self-Association and Its Role in 
            Condensate Formation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6046)
  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            "Unpublished"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_549-638 (RGG3)
FEATURES             Location/Qualifiers
     source          1..6046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(130..148)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      144..191
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(366..371,372..388,389..394))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(421..439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        507..611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(612..680,681..1472)
                     /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(830..849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1643..2231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2132..2151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2385..2402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2417..2556
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2482..2501)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2642..2664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2658..2849)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4551..4570
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4551..4569
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4570..4594
                     /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             4609..4631
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4644..4673
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4680..5780
                     /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     5754..5777
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5829..5849
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 tcgtcgtggc ggtccgggcg gtccgccggg tccgctgatg gaacagatgg gcggtcgtcg
       61 tggcggtcgt ggcggttaag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      121 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      181 ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac
      241 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
      301 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
      361 accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg
      421 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
      481 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
      541 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      601 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      661 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      721 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      781 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      841 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      901 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      961 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1021 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1081 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1141 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1201 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1261 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1321 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1381 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     1441 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1501 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1561 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1621 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1681 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1741 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     1801 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1861 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1921 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1981 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2041 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2101 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2161 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2221 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2281 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2341 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2401 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2461 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     2521 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     2581 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2641 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     2701 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     2761 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     2821 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     2881 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     2941 gaacatgccc ggttactgga acgtcgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3001 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3061 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3121 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3181 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3241 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3301 atcatgcaca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     3361 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     3421 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     3481 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     3541 caaggtatag ggcggcacct acaatccatg acaaggtata gggcggcacc tacaatccat
     3601 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     3661 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     3721 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     3781 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     3841 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3901 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3961 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4021 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4081 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4141 tgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     4201 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggagcccaa cagtcccccc
     4261 atgcaaggag atggagccca acagtcccca tgcaaggaga tggagcccaa cagtcccacg
     4321 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4381 gcccgatctt ccccatcggt gatgtcggcg atataggcgc aagcaaccgc acctgtagcg
     4441 ccggtgatgc cgggtcggcg atataggcgc aagcaaccgc acctgtagcg ccggtgatgc
     4501 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     4561 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     4621 taagaaggag atatcatatg ggccatcatc atcatcatca tcatcatcat cacagcagca
     4681 tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
     4741 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
     4801 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     4861 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     4921 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     4981 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     5041 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     5101 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     5161 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     5221 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     5281 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
     5341 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     5401 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     5461 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     5521 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     5581 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     5641 ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     5701 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     5761 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca
     5821 acctcgggga gaatctttat tttcagggcg ccgaaggttt cctgccgccg ccgtttccgc
     5881 cgccgggtgg tgaccgtggt cgtggtggcc cgggtggcat gcgtggtggc cgtggtggcc
     5941 tgatggaccg tggtggcccg ggtggcatgt ttcgtggtgg ccgtggtggc gaccgtggtg
     6001 gctttcgtgg tggccgtggc atggatcgtg gtggcttcgg tggcgg
//
