LOCUS       sequence-392954-        7228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7228)
  AUTHORS   Sabri N, Cuneo MJ, Marzahn MR, Lee J, Bouchard JJ, Gullulu O, 
            Vaithiyalingam S, Borgia MB, Schmit J, Mittag T
  TITLE     Reduction of oligomer size modulates the competition between cluster
            formation and phase separation of the tumor suppressor SPOP.
  JOURNAL   J Biol Chem. 2023 Dec;299(12):105427. doi: 
            10.1016/j.jbc.2023.105427. Epub 2023 Nov 4.
  PUBMED    37926283
REFERENCE   2  (bases 1 to 7228)
  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: "J Biol 
            Chem."; date: "2023-12"; volume: "299(12):105427. doi"; pages: " 
            10.1016/j.jbc.2023.105427. Epub 2023 Nov 4"
COMMENT     Sequence Label: His SUMO TEV R354H SPOP 28-359
FEATURES             Location/Qualifiers
     source          1..7228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1850..1869
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1889..1966)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2172..2191
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2275..2294
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2275..2293
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2294..2318
                     /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             2333..2355
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2375..2392
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2402..2419
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2426..2719
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    2726..2750
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2753..2773
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(3903..3927)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4004..4022)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4018..4065
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4240..4245,4246..4262,4263..4268))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4295..4313)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4381..4485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4486..4554,4555..5346)
                     /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(4704..4723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5517..6105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6006..6025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6259..6276
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6291..6430
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6356..6375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6516..6538
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6532..6723)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
       61 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
      121 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
      181 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
      241 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
      301 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
      361 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
      421 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
      481 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
      541 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
      601 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
      661 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
      721 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
      781 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aactgtcgtg
      841 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
      901 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
      961 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     1021 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     1081 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     1141 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     1201 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     1261 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     1321 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     1381 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     1441 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     1501 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     1561 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     1621 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     1681 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     1741 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     1801 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     1861 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1921 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1981 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     2041 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     2101 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     2161 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     2221 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     2281 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     2341 actttaagaa ggagatatac atatgggcag cagccatcat catcatcatc acggcagcgg
     2401 cctggtgccg cgcggcagcg ctagcatgtc ggactcagaa gtcaatcaag aagctaagcc
     2461 agaggtcaag ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatggatc
     2521 ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt
     2581 cgctaaaaga cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat
     2641 tcaagctgat cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca
     2701 cagagaacag attggtggta gtatcacaag tttgtacaaa aaagcaggct tcgaaaacct
     2761 gtattttcag ggcagcatgt caagggttcc aagtcctcca cctccggcag aaatgtcgag
     2821 tggtcccgtt gccgagagct ggtgctatac acagatcaag gtagtgaagt tctcctacat
     2881 gtggaccatc aataacttta gtttttgccg ggaggaaatg ggtgaagtca taaaaagttc
     2941 aactttttca tcaggagcca atgataaact gaaatggtgt ttgcgagtaa acccgaaagg
     3001 gctagatgaa gaaagcaaag attacctgtc gctttacctg ttgttggtca gctgtccaaa
     3061 gagtgaagtt cgggcaaagt tcaagttctc catcctgaat gccaagggag aagaaaccaa
     3121 agctatggag agtcagcgag cttataggtt cgtgcaaggc aaagactggg gattcaaaaa
     3181 attcatccgc agagattttc tcttggacga ggccaatggg cttctccctg atgacaagct
     3241 caccctcttc tgtgaggtga gtgtagtaca agactctgtc aatatttctg ggcagaatac
     3301 tatgaacatg gtaaaggttc ctgaatgccg gttggcagat gagttaggag ggctgtggga
     3361 gaactctcga ttcaccgact gctgcttgtg tgtggctggt caagaattcc aggctcacaa
     3421 ggctatctta gcagctcggt ctccagtctt cagtgccatg tttgaacatg aaatggagga
     3481 gagcaaaaag aatcgggttg agattaacga cgtggagcct gaggtgttta aggagatgat
     3541 gtgcttcatc tacacgggga aggcgccgaa cctcgacaag atggctgacg acttgctggc
     3601 agctgccgat aagtacgccc tggagcgtct gaaggtcatg tgtgaggacg ccctctgcag
     3661 taacctctct gtggagaacg ctgcagagat tctcatcctg gctgacctcc acagcgcaga
     3721 tcagctgaag acgcaggcag ttgatttcat caactatcat gcttcggatg tcttggagac
     3781 ctctgggtgg aagtcaatgg tggtgtcaca tccccactta gtggcagaag cataccattc
     3841 tctggcttca gcacagtgcc ctttcctggg gcccccacgc aaacgcctga agcaatccta
     3901 ggacccagct ttcttgtaca aagtggtgat actggatcca tgggtaccct cgaggatccg
     3961 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     4021 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     4081 atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     4141 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     4201 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa
     4261 gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     4321 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4381 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4441 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4501 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4561 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4621 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4681 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     4741 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4801 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4861 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4921 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4981 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     5041 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5101 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5161 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5221 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5281 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5341 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5401 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5461 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5521 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5581 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5641 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5701 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5761 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5821 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5881 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5941 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6001 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6061 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6121 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6181 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6241 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     6301 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     6361 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     6421 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6481 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6541 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     6601 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     6661 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     6721 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     6781 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     6841 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     6901 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcccgc
     6961 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     7021 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     7081 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     7141 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
     7201 aacgctgccc gagatgcgcc gcgtgcgg
//
