LOCUS       sequence-389538-        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  E47K SPOP 1-374
FEATURES             Location/Qualifiers
     source          1..7228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     960..979
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(999..1076)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1282..1301
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1385..1404
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1385..1403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1404..1428
                     /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             1443..1465
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1485..1502
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1512..1529
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1536..1829
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    1836..1860
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1863..1883
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(3013..3037)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3114..3132)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3128..3175
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3350..3355,3356..3372,3373..3378))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3405..3423)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3491..3595
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3596..3664,3665..4456)
                     /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(3814..3833)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4627..5215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5116..5135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5369..5386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5401..5540
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5466..5485)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5626..5648
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5642..5833)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
       61 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      121 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      181 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      241 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      301 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      361 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      421 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      481 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      541 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
      601 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
      661 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
      721 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
      781 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
      841 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
      901 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
      961 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1021 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1081 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1141 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1201 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     1261 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1321 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     1381 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     1441 attttgttta actttaagaa ggagatatac atatgggcag cagccatcat catcatcatc
     1501 acggcagcgg cctggtgccg cgcggcagcg ctagcatgtc ggactcagaa gtcaatcaag
     1561 aagctaagcc agaggtcaag ccagaagtca agcctgagac tcacatcaat ttaaaggtgt
     1621 ccgatggatc ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctga
     1681 tggaagcgtt cgctaaaaga cagggtaagg aaatggactc cttaagattc ttgtacgacg
     1741 gtattagaat tcaagctgat cagacccctg aagatttgga catggaggat aacgatatta
     1801 ttgaggctca cagagaacag attggtggta gtatcacaag tttgtacaaa aaagcaggct
     1861 tcgaaaacct gtattttcag ggcagcatgt caagggttcc aagtcctcca cctccggcag
     1921 aaatgtcgag tggtcccgtt gccgagagct ggtgctatac acagatcaag gtagtgaagt
     1981 tctcctacat gtggaccatc aataacttta gtttttgccg ggagaaaatg ggtgaagtca
     2041 taaaaagttc aactttttca tcaggagcca atgataaact gaaatggtgt ttgcgagtaa
     2101 acccgaaagg gctagatgaa gaaagcaaag attacctgtc gctttacctg ttgttggtca
     2161 gctgtccaaa gagtgaagtt cgggcaaagt tcaagttctc catcctgaat gccaagggag
     2221 aagaaaccaa agctatggag agtcagcgag cttataggtt cgtgcaaggc aaagactggg
     2281 gattcaaaaa attcatccgc agagattttc tcttggacga ggccaatggg cttctccctg
     2341 atgacaagct caccctcttc tgtgaggtga gtgtagtaca agactctgtc aatatttctg
     2401 ggcagaatac tatgaacatg gtaaaggttc ctgaatgccg gttggcagat gagttaggag
     2461 ggctgtggga gaactctcga ttcaccgact gctgcttgtg tgtggctggt caagaattcc
     2521 aggctcacaa ggctatctta gcagctcggt ctccagtctt cagtgccatg tttgaacatg
     2581 aaatggagga gagcaaaaag aatcgggttg agattaacga cgtggagcct gaggtgttta
     2641 aggagatgat gtgcttcatc tacacgggga aggcgccgaa cctcgacaag atggctgacg
     2701 acttgctggc agctgccgat aagtacgccc tggagcgtct gaaggtcatg tgtgaggacg
     2761 ccctctgcag taacctctct gtggagaacg ctgcagagat tctcatcctg gctgacctcc
     2821 acagcgcaga tcagctgaag acgcaggcag ttgatttcat caactatcat gcttcggatg
     2881 tcttggagac ctctgggtgg aagtcaatgg tggtgtcaca tccccactta gtggcagaag
     2941 cataccgttc tctggcttca gcacagtgcc ctttcctggg gcccccacgc aaacgcctga
     3001 agcaatccta ggacccagct ttcttgtaca aagtggtgat actggatcca tgggtaccct
     3061 cgaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     3121 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     3181 aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct
     3241 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc
     3301 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta
     3361 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct
     3421 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3481 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3541 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3601 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3661 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3721 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3781 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     3841 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3901 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3961 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4021 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4081 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     4141 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4201 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4261 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4321 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4381 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4441 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4501 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4561 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4621 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4681 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4741 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4801 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4861 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4921 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4981 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5041 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5101 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5161 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5221 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5281 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5341 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5401 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     5461 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     5521 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5581 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5641 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     5701 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5761 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5821 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5881 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5941 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     6001 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     6061 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     6121 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     6181 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     6241 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     6301 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     6361 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     6421 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     6481 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     6541 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     6601 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     6661 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     6721 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     6781 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     6841 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     6901 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     6961 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     7021 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     7081 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     7141 gcgctcactg cccgctttcc agtcgggaaa ctgtcgtgcc agctgcatta atgaatcggc
     7201 caacgcgcgg ggagaggcgg tttgcgta
//
