LOCUS       sequence-359559-        5681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the construct His8-GFP-EWS_1-120 with a TEV cleavage site 
            between GFP and EWS_1-120.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  AUTHORS   Johnson CN, Xu X, Holloway SP, Libich DS
  TITLE     The (1)H, (15)N and (13)C resonance assignments of the 
            low-complexity domain from the oncogenic fusion protein EWS-FLI1.
  JOURNAL   Biomol NMR Assign. 2022 Apr;16(1):67-73. doi: 
            10.1007/s12104-021-10061-4. Epub 2022 Jan 7.
  PUBMED    34994941
REFERENCE   2  (bases 1 to 5681)
  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"; doi: 
            "10.1007/s12104-021-10061-4"; journalName: "Biomol NMR Assign"; 
            date: "2022-04"; volume: "16"; issue: "1"; pages: "67-73"
COMMENT     Sequence Label: pAG8H-GFP-EWS_1-120
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             74..96
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             111..134
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             141..848
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITLGMENLY"
     primer_bind     complement(180..201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     788..809
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             837..857
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1283..1301)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1297..1344
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1519..1524,1525..1542,1543..1548))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1575..1593)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1661..1765
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1766..1834,1835..2626)
                     /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(1984..2003)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2797..3385
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3286..3305
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3539..3556
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3571..3710
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3636..3655)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3796..3818
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3812..4003)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc
       61 ctctagaaat aattttgttt aactttaaga aggagatata ccatatggac caccatcacc
      121 accaccatca ccatgctagc agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      181 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg
      241 gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      301 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      361 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      421 agcgcaccat ctccttcaag gacgacggca cctacaagac ccgcgccgag gtgaagttcg
      481 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      541 acatcctggg gcacaagctg gagtacaact tcaacagcca caacgtctat atcacggccg
      601 acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacgtc gaggacggca
      661 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      721 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
      781 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggaga
      841 atctgtactt tcagggcgcc agcaccgatt atagcaccta tagccaggcc gcggcccagc
      901 agggttatag cgcctatacc gcgcagccga cccagggtta tgcccagacc acccaggcgt
      961 atggccagca gagctatggt acctatggcc agccgaccga tgtgagctat acccaggccc
     1021 agaccaccgc gacctatggt cagaccgcct atgccaccag ctatggtcag ccgccgaccg
     1081 gttataccac cccgaccgcc ccgcaggcct atagccagcc ggttcagggc tatggtaccg
     1141 gtgcctatga taccaccacc gccaccgtga ccaccaccca ggcaagctat gcggcccaga
     1201 gcgcctatgg tacctagcag cagtgaggtc tcggatccgg ctgctaacaa agcccgaaag
     1261 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1321 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata tcccgcaaga
     1381 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
     1441 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     1501 actgtgataa actaccgcat taaagcttat cggatgataa gctgtcaaac atgagaattc
     1561 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     1621 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1681 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1741 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1801 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1861 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1921 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1981 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     2041 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2101 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2161 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2221 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2281 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2341 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2401 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2461 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2521 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2581 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2641 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2701 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2761 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2821 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2881 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2941 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3001 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3061 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3121 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3181 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3241 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3301 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3361 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3421 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3481 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3541 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3601 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3661 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3721 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3781 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3841 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3901 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3961 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4021 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4081 gggttactga tgatgaacat gcccggttac tggaacgtcg tgagggtaaa caactggcgg
     4141 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4201 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4261 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4321 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4381 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4441 acgacaggag cacgatcatg cacacccgtg gccaggaccc aacgctgccc gagatgcgcc
     4501 gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg
     4561 cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag
     4621 cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa
     4681 cgcggggagg cagacaaggt atagggcggc acctacaatc catgacaagg tatagggcgg
     4741 cacctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga
     4801 cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
     4861 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga
     4921 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg
     4981 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
     5041 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5101 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5161 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5221 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5281 tcaagggcat cggttgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5341 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggagc
     5401 ccaacagtcc ccccatgcaa ggagatggag cccaacagtc cccatgcaag gagatggagc
     5461 ccaacagtcc cacggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5521 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgcaagcaa
     5581 ccgcacctgt agcgccggtg atgccgggtc ggcgatatag gcgcaagcaa ccgcacctgt
     5641 agcgccggtg atgccggcca cgatgcgtcc ggcgtagagg a
//
