LOCUS       sequence-390432-        7228 bp    DNA     circular SYN 15-APR-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 Apr 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  W131G SPOP 1-374
FEATURES             Location/Qualifiers
     source          1..7228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2401..2420
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2440..2517)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2723..2742
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2826..2845
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2826..2844
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2845..2869
                     /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             2884..2906
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2926..2943
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2953..2970
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2977..3270
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     protein_bind    3277..3301
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3304..3324
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(4454..4478)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4555..4573)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4569..4616
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4791..4796,4797..4813,4814..4819))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4846..4864)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4932..5036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5037..5105,5106..5897)
                     /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(5255..5274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6068..6656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6557..6576
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6810..6827
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6842..6981
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6907..6926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7067..7089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(7083..7228,1..46))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
       61 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
      121 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
      181 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
      241 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
      301 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
      361 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
      421 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
      481 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
      541 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
      601 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
      661 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
      721 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
      781 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
      841 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
      901 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
      961 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     1021 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     1081 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     1141 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     1201 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     1261 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     1321 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1381 aaactgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1441 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     1501 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     1561 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     1621 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     1681 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     1741 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     1801 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     1861 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     1921 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     1981 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     2041 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     2101 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     2161 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     2221 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     2281 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     2341 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     2401 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     2461 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     2521 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     2581 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     2641 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     2701 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     2761 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     2821 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     2881 aattttgttt aactttaaga aggagatata catatgggca gcagccatca tcatcatcat
     2941 cacggcagcg gcctggtgcc gcgcggcagc gctagcatgt cggactcaga agtcaatcaa
     3001 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     3061 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     3121 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     3181 ggtattagaa ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     3241 attgaggctc acagagaaca gattggtggt agtatcacaa gtttgtacaa aaaagcaggc
     3301 ttcgaaaacc tgtattttca gggcagcatg tcaagggttc caagtcctcc acctccggca
     3361 gaaatgtcga gtggtcccgt tgccgagagc tggtgctata cacagatcaa ggtagtgaag
     3421 ttctcctaca tgtggaccat caataacttt agtttttgcc gggaggaaat gggtgaagtc
     3481 ataaaaagtt caactttttc atcaggagcc aatgataaac tgaaatggtg tttgcgagta
     3541 aacccgaaag ggctagatga agaaagcaaa gattacctgt cgctttacct gttgttggtc
     3601 agctgtccaa agagtgaagt tcgggcaaag ttcaagttct ccatcctgaa tgccaaggga
     3661 gaagaaacca aagctatgga gagtcagcga gcttataggt tcgtgcaagg caaagacggg
     3721 ggattcaaaa aattcatccg cagagatttt ctcttggacg aggccaatgg gcttctccct
     3781 gatgacaagc tcaccctctt ctgtgaggtg agtgtagtac aagactctgt caatatttct
     3841 gggcagaata ctatgaacat ggtaaaggtt cctgaatgcc ggttggcaga tgagttagga
     3901 gggctgtggg agaactctcg attcaccgac tgctgcttgt gtgtggctgg tcaagaattc
     3961 caggctcaca aggctatctt agcagctcgg tctccagtct tcagtgccat gtttgaacat
     4021 gaaatggagg agagcaaaaa gaatcgggtt gagattaacg acgtggagcc tgaggtgttt
     4081 aaggagatga tgtgcttcat ctacacgggg aaggcgccga acctcgacaa gatggctgac
     4141 gacttgctgg cagctgccga taagtacgcc ctggagcgtc tgaaggtcat gtgtgaggac
     4201 gccctctgca gtaacctctc tgtggagaac gctgcagaga ttctcatcct ggctgacctc
     4261 cacagcgcag atcagctgaa gacgcaggca gttgatttca tcaactatca tgcttcggat
     4321 gtcttggaga cctctgggtg gaagtcaatg gtggtgtcac atccccactt agtggcagaa
     4381 gcataccgtt ctctggcttc agcacagtgc cctttcctgg ggcccccacg caaacgcctg
     4441 aagcaatcct aggacccagc tttcttgtac aaagtggtga tactggatcc atgggtaccc
     4501 tcgaggatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     4561 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     4621 aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca taaccaagcc
     4681 tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt
     4741 catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt
     4801 atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc
     4861 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4921 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4981 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5041 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5101 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5161 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5221 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     5281 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5341 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5401 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5461 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5521 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5581 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5641 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5701 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5761 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5821 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5881 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5941 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6001 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6061 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6121 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6181 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     6241 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6301 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6361 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6421 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6481 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6541 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6601 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6661 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6721 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6781 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6841 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     6901 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     6961 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     7021 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     7081 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     7141 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     7201 ttctccagaa gcgttaatgt ctggcttc
//
