LOCUS       sequence-401659-        5971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RG2 (the second RG-rich region, 449-513) of EWS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5971)
  AUTHORS   Selig EE, Bhura R, White MR, Akula S, Hoffman RD, Tovar CN, Xu X, 
            Booth RE, Libich DS
  TITLE     Biochemical and biophysical characterization of the nucleic acid 
            binding properties of the RNA/DNA binding protein EWS.
  JOURNAL   Biopolymers. 2023 May;114(5):e23536. doi: 10.1002/bip.23536. Epub 
            2023 Mar 17.
  PUBMED    36929870
REFERENCE   2  (bases 1 to 5971)
  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: 
            "Biopolymers."; date: "2023-05"; volume: "114(5):e23536. doi"; 
            pages: " 10.1002/bip.23536. Epub 2023 Mar 17"
COMMENT     Sequence Label: pEmal10H-MBP-EWSR1-449-513
FEATURES             Location/Qualifiers
     source          1..5971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1681..1700
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1681..1699
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1700..1724
                     /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             1739..1761
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1774..1803
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1810..2910
                     /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     2884..2907
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2959..2979
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3231..3249)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3245..3292
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3467..3472,3473..3489,3490..3495))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3522..3540)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3608..3712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3713..3781,3782..4573)
                     /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(3931..3950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4744..5332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5233..5252
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5486..5503
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5518..5657
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5583..5602)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5743..5765
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5759..5950)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
       61 tactgatgat gaacatgccc ggttactgga acgtcgtgag ggtaaacaac tggcggtatg
      121 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
      181 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
      241 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
      301 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
      361 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
      421 caggagcacg atcatgcaca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt
      481 gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt
      541 cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag
      601 gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg
      661 gggaggcaga caaggtatag ggcggcacct acaatccatg acaaggtata gggcggcacc
      721 tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat
      781 cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc
      841 ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc
      901 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag
      961 caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa
     1021 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     1081 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     1141 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     1201 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     1261 gggcatcggt tgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     1321 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggagcccaa
     1381 cagtcccccc atgcaaggag atggagccca acagtcccca tgcaaggaga tggagcccaa
     1441 cagtcccacg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     1501 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc aagcaaccgc
     1561 acctgtagcg ccggtgatgc cgggtcggcg atataggcgc aagcaaccgc acctgtagcg
     1621 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1681 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1741 tgtttaactt taagaaggag atatcatatg ggccatcatc atcatcatca tcatcatcat
     1801 cacagcagca tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
     1861 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
     1921 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
     1981 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
     2041 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
     2101 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
     2161 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
     2221 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
     2281 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
     2341 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
     2401 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
     2461 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
     2521 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
     2581 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     2641 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     2701 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     2761 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     2821 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     2881 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac
     2941 aataacaaca acctcgggga gaatctttat tttcagggcg ccccgccgat gaacagcatg
     3001 cgtggcggtc tgccgccgcg tgagggtcgt ggcatgccgc cgccactgcg tggcggtccg
     3061 ggcggtccgg gcggtccggg cggtccgatg ggtcgtatgg gcggtcgtgg cggtgaccgt
     3121 ggcggttttc cgccgcgtgg tccgcgtggc agccgtggta acccgagcgg cggtggctga
     3181 ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3241 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     3301 aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     3361 cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata
     3421 cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg
     3481 atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt
     3541 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3601 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3661 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3721 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3781 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3841 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3901 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     3961 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4021 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4081 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4141 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     4201 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     4261 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4321 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4381 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4441 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4501 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4561 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4621 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4681 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4741 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4801 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4861 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     4921 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4981 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5041 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5101 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5161 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5221 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5281 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5341 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5401 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5461 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     5521 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     5581 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     5641 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5701 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5761 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     5821 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     5881 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     5941 gtttggtcac tgatgcctcc gtgtaagggg g
//
