LOCUS       sequence-389539-        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   Cuneo MJ, O'Flynn BG, Lo YH, Sabri N, Mittag T
  TITLE     Higher-order SPOP assembly reveals a basis for cancer mutant 
            dysregulation.
  JOURNAL   Mol Cell. 2023 Mar 2;83(5):731-745.e4. doi: 
            10.1016/j.molcel.2022.12.033. Epub 2023 Jan 23.
  PUBMED    36693379
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"; doi: 
            "10.1016/j.molcel.2022.12.033"; journalName: "Mol Cell"; date: 
            "2023-03-2- 2"; volume: "83"; issue: "5"; pages: "731-745.e4"
COMMENT     Sequence Label: His SUMO TEV  E78K SPOP 1-374
FEATURES             Location/Qualifiers
     source          1..7228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     554..573
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(593..670)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     876..895
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     979..998
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        979..997
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    998..1022
                     /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             1037..1059
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1079..1096
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1106..1123
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1130..1423
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    1430..1454
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1457..1477
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(2607..2631)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2708..2726)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2722..2769
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2944..2949,2950..2966,2967..2972))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2999..3017)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3085..3189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3190..3258,3259..4050)
                     /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(3408..3427)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4221..4809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4710..4729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4963..4980
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4995..5134
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5060..5079)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5220..5242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5236..5427)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
       61 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
      121 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
      181 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
      241 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
      301 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
      361 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
      421 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
      481 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
      541 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
      601 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
      661 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
      721 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
      781 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
      841 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
      901 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
      961 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1021 cccctctaga aataattttg tttaacttta agaaggagat atacatatgg gcagcagcca
     1081 tcatcatcat catcacggca gcggcctggt gccgcgcggc agcgctagca tgtcggactc
     1141 agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat
     1201 caatttaaag gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc
     1261 tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag
     1321 attcttgtac gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga
     1381 ggataacgat attattgagg ctcacagaga acagattggt ggtagtatca caagtttgta
     1441 caaaaaagca ggcttcgaaa acctgtattt tcagggcagc atgtcaaggg ttccaagtcc
     1501 tccacctccg gcagaaatgt cgagtggtcc cgttgccgag agctggtgct atacacagat
     1561 caaggtagtg aagttctcct acatgtggac catcaataac tttagttttt gccgggagga
     1621 aatgggtgaa gtcataaaaa gttcaacttt ttcatcagga gccaatgata aactgaaatg
     1681 gtgtttgcga gtaaacccga aagggctaga taaagaaagc aaagattacc tgtcgcttta
     1741 cctgttgttg gtcagctgtc caaagagtga agttcgggca aagttcaagt tctccatcct
     1801 gaatgccaag ggagaagaaa ccaaagctat ggagagtcag cgagcttata ggttcgtgca
     1861 aggcaaagac tggggattca aaaaattcat ccgcagagat tttctcttgg acgaggccaa
     1921 tgggcttctc cctgatgaca agctcaccct cttctgtgag gtgagtgtag tacaagactc
     1981 tgtcaatatt tctgggcaga atactatgaa catggtaaag gttcctgaat gccggttggc
     2041 agatgagtta ggagggctgt gggagaactc tcgattcacc gactgctgct tgtgtgtggc
     2101 tggtcaagaa ttccaggctc acaaggctat cttagcagct cggtctccag tcttcagtgc
     2161 catgtttgaa catgaaatgg aggagagcaa aaagaatcgg gttgagatta acgacgtgga
     2221 gcctgaggtg tttaaggaga tgatgtgctt catctacacg gggaaggcgc cgaacctcga
     2281 caagatggct gacgacttgc tggcagctgc cgataagtac gccctggagc gtctgaaggt
     2341 catgtgtgag gacgccctct gcagtaacct ctctgtggag aacgctgcag agattctcat
     2401 cctggctgac ctccacagcg cagatcagct gaagacgcag gcagttgatt tcatcaacta
     2461 tcatgcttcg gatgtcttgg agacctctgg gtggaagtca atggtggtgt cacatcccca
     2521 cttagtggca gaagcatacc gttctctggc ttcagcacag tgccctttcc tggggccccc
     2581 acgcaaacgc ctgaagcaat cctaggaccc agctttcttg tacaaagtgg tgatactgga
     2641 tccatgggta ccctcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2701 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2761 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
     2821 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     2881 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     2941 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     3001 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     3061 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3121 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3181 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3241 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3301 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3361 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3421 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3481 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3541 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3601 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3661 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3721 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3781 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3841 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3901 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3961 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4021 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4081 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4141 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4201 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4261 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4321 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4381 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4441 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4501 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4561 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4621 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4681 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4741 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4801 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4861 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4921 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4981 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5041 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5101 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5161 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5221 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5281 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5341 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5401 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5461 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5521 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5581 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5641 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5701 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5761 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5821 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5881 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     5941 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     6001 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     6061 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     6121 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     6181 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     6241 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     6301 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     6361 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     6421 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     6481 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     6541 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     6601 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     6661 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     6721 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaactgtcg tgccagctgc
     6781 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     6841 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6901 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6961 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     7021 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     7081 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     7141 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     7201 cgagtgagat atttatgcca gccagcca
//
