LOCUS       sequence-382419-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  R221C His SUMO SPOP expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7102)
  AUTHORS   Thomasen FE, Cuneo MJ, Mittag T, Lindorff-Larsen K
  TITLE     Conformational and oligomeric states of SPOP from small-angle X-ray 
            scattering and molecular dynamics simulations.
  JOURNAL   Elife. 2023 Mar 1;12:e84147. doi: 10.7554/eLife.84147.
  PUBMED    36856266
REFERENCE   2  (bases 1 to 7102)
  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: "Elife."; 
            date: "2023-03-1"; volume: "12:e84147. doi"; pages: " 
            10.7554/eLife.84147"
COMMENT     Sequence Label: His SUMO TEV R221C SPOP 28-359
FEATURES             Location/Qualifiers
     source          1..7102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..76
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        57..75
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    76..100
                     /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             115..137
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             157..174
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             184..201
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             208..501
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    508..532
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             535..555
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(1559..1583)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1660..1678)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1674..1721
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1896..1901,1902..1918,1919..1924))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1951..1969)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2037..2141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2142..2210,2211..3002)
                     /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(2360..2379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3173..3761
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3662..3681
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3915..3932
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3947..4086
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4012..4031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4172..4194
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4188..4379)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6734..6753
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6773..6850)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7056..7075
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
       61 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      121 taactttaag aaggagatat acatatgggc agcagccatc atcatcatca tcacggcagc
      181 ggcctggtgc cgcgcggcag cgctagcatg tcggactcag aagtcaatca agaagctaag
      241 ccagaggtca agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga
      301 tcttcagaga tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg
      361 ttcgctaaaa gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga
      421 attcaagctg atcagacccc tgaagatttg gacatggagg ataacgatat tattgaggct
      481 cacagagaac agattggtgg tagtatcaca agtttgtaca aaaaagcagg cttcgaaaac
      541 ctgtattttc agggcagcaa ggtagtgaaa ttctcctaca tgtggaccat caataacttt
      601 agcttttgcc gggaggaaat gggtgaagtc attaaaagtt ctacattttc atcaggagca
      661 aatgataaac tgaaatggtg tttgcgagta aaccccaaag ggttagatga agaaagcaaa
      721 gattacctgt cactttacct gttactggtc agctgtccaa agagtgaagt tcgggcaaaa
      781 ttcaaattct ccatcctgaa tgccaaggga gaagaaacca aagctatgga gagtcaacgg
      841 gcatataggt ttgtgcaagg caaagactgg ggattcaaga aattcatccg tagagatttt
      901 cttttggatg aggccaacgg gcttctcccc gatgacaagc ttaccctctt ctgcgaggtg
      961 agtgttgtgc aagattctgt caacatttct ggccagaata ccatgaacat ggtaaaggtt
     1021 cctgagtgcc ggctggcaga tgagttagga ggactgtggg agaattcccg gttcacagac
     1081 tgctgcttgt gtgttgccgg ccaggaattc caggctcaca aggctatctt agcagcttgt
     1141 tctccggttt ttagtgccat gtttgaacat gaaatggagg agagcaaaaa gaatcgagtt
     1201 gaaatcaatg atgtggagcc tgaagttttt aaggaaatga tgtgcttcat ttacacgggg
     1261 aaggctccaa acctcgacaa aatggctgat gatttgctgg cagctgctga caagtatgcc
     1321 ctggagcgct taaaggtcat gtgtgaggat gccctctgca gtaacctgtc cgtggagaac
     1381 gctgcagaaa ttctcatcct ggccgacctc cacagtgcag atcagttgaa aactcaggca
     1441 gtggatttca tcaactatca tgcttcggat gtcttggaga cctctgggtg gaagtcaatg
     1501 gtggtgtcac atccccactt ggtggctgag gcataccgct ctctggcttc agcataaaac
     1561 ccagctttct tgtacaaagt ggtgatactg gatccatggg taccctcgag gatccggctg
     1621 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1681 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1741 ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     1801 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     1861 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     1921 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1981 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     2041 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2101 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2161 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2221 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2281 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2341 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     2401 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2461 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2521 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2581 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2641 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2701 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2761 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2821 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2881 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2941 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3001 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3061 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3181 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3241 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3301 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3361 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3421 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3481 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3541 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3601 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3661 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3721 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3781 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3841 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3901 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3961 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     4021 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4081 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4141 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4201 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     4261 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     4321 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     4381 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4441 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4501 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4561 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4621 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4681 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4741 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4801 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     4861 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
     4921 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
     4981 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
     5041 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
     5101 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     5161 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     5221 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     5281 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     5341 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     5401 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     5461 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     5521 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     5581 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     5641 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     5701 ctttccagtc gggaaactgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5761 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5821 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5881 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5941 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     6001 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     6061 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     6121 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     6181 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     6241 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     6301 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     6361 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     6421 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6481 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6541 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6601 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6661 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6721 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6781 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6841 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     6901 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     6961 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     7021 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     7081 gcaaccgcac ctgtggcgcc gg
//
