LOCUS       sequence-376804-        4883 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses His8-EWS_RRM(359-447) 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 4883)
  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 4883)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pAG8Ha-EWS_RRM(359-447)
FEATURES             Location/Qualifiers
     source          1..4883
                     /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(599..619)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(626..649)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     RBS             664..686
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(717..736)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(718..736)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2438..2629
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2623..2645)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2731..2870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2786..2805
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2885..2902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3056..3644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3136..3155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3815..4606,4607..4675))
                     /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     4438..4457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4676..4780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4848..4866
                     /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 gatcctcact tacgcgccag gctcactttc agcttgctac
      361 cctgaaaatc tttgccgtcg aaccattcaa ccgccgcctt cgcggtcggc gggtcctcgt
      421 agctcacggt cgcgtcacct ttcggcttgc cggtttcttt atccaggtaa atgtggatca
      481 tcggttgacc ggtacgtttg ttcatcttaa ccacgccgca ctgcttaaag aaatccgcca
      541 gatcgtccag ggtaacgctg tcgttcagac cctgcacgta gatcgcgctg ttatcggcgc
      601 cctgaaaata aagattctcg ctagcatggt gatggtggtg gtgatggtgg tccatatggt
      661 atatctcctt cttaaagtta aacaaaatta tttctagagg gaaaccgttg tggtctccct
      721 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
      781 tggccggcat caccggcgct acaggtgcgg ttgcttgcgc ctatatcgcc gacccggcat
      841 caccggcgct acaggtgcgg ttgcttgcgc ctatatcgcc gacatcaccg atggggaaga
      901 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
      961 cgtggccgtg ggactgttgg gctccatctc cttgcatggg gactgttggg ctccatctcc
     1021 ttgcatgggg ggactgttgg gctccatctc cttgcatgca ccattccttg cggcggcggt
     1081 gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga
     1141 gcgtcaaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     1201 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     1261 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     1321 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     1381 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc
     1441 gctgggctac gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat
     1501 tcttctcgct tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt
     1561 agatgacgac catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc
     1621 gatcattgga ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg
     1681 gttggcatgg attgtaggtg ccgccctata ccttgtcatg gattgtaggt gccgccctat
     1741 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa
     1801 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt
     1861 cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc
     1921 catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgtg
     1981 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     2041 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     2101 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     2161 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     2221 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     2281 ctggtcccgc cgcatccata ccgccagttg tttaccctca cgacgttcca gtaaccgggc
     2341 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     2401 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     2461 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     2521 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     2581 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2641 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2701 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     2761 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     2821 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     2881 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2941 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3001 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3061 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3121 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3181 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3241 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3301 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3361 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3421 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3481 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3541 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3601 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3661 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3721 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3781 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3841 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3901 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3961 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4021 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4081 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg
     4141 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4201 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4261 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4321 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4381 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     4441 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4501 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4561 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4621 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4681 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4741 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4801 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4861 cgaggccctt tcgtcttcaa gaa
//
