LOCUS       sequence-394909-        6852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid expresses Flag-tagged FBW7 human isoform 1 with Lys404 
            to Arg mutation. Used for mammalian overexpression and detection by 
            western blotting..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6852)
  AUTHORS   Khan OM, Almagro J, Nelson JK, Horswell S, Encheva V, Keyan KS, 
            Clurman BE, Snijders AP, Behrens A
  TITLE     Proteasomal degradation of the tumour suppressor FBW7 requires 
            branched ubiquitylation by TRIP12.
  JOURNAL   Nat Commun. 2021 Apr 6;12(1):2043. doi: 10.1038/s41467-021-22319-5.
  PUBMED    33824312
REFERENCE   2  (bases 1 to 6852)
  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: "Nat 
            Commun."; date: "2021-04-6"; volume: "12(1):2043. doi"; pages: " 
            10.1038/s41467-021-22319-5"
COMMENT     Sequence Label: pDEST-Flag-FBW7K404R
FEATURES             Location/Qualifiers
     source          1..6852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..465
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        466..669
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     620..640
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             699..764
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    768..792
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2917..2941)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2961..3583
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3160..3179)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3607..3627)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3612..3941
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3792..3927
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3979..3998)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3980..3998)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4012..4028)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4012..4028)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4025..4047)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4036..4052
                     /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)."
     promoter        complement(join(4060..4066,4067..4084,4085..4090))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4105..4126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4243..4260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4414..5002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4494..4513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5173..5964,5965..6033))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5796..5815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6034..6138)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6165..6620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6252..6271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6462..6483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6746..6768
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6760..6777
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6761..6777
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc
       61 tcatgtccaa tatgaccgcc atgttgacat tgattattga ctagttatta atagtaatca
      121 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      181 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      241 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      301 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac
      361 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt
      421 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      481 cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      541 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      601 aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      661 agcagagctc gtttagtgaa ccgtcagaat tcaccatgga ctacaaagac catgacggtg
      721 attataaaga tcatgatatc gattacaagg atgacgatga caagcttaca agtttgtaca
      781 aaaaagcagg ctttaatcag gaactgctct ctgtgggcag caaaagacga cgaactggag
      841 gctctctgag aggtaaccct tcctcaagcc aggtagatga agaacagatg aatcgtgtgg
      901 tagaggagga acagcaacag caactcagac aacaagagga ggagcacact gcaaggaatg
      961 gtgaagttgt tggagtagaa cctagacctg gaggccaaaa tgattcccag caaggacagt
     1021 tggaagaaaa caataataga tttatttcgg tagatgagga ctcctcagga aaccaagaag
     1081 aacaagagga agatgaagaa catgctggtg aacaagatga ggaggatgag gaggaggagg
     1141 agatggacca ggagagtgac gattttgatc agtctgatga tagtagcaga gaagatgaac
     1201 atacacatac taacagtgtc acgaactcca gtagtattgt ggacctgccc gttcaccaac
     1261 tctcctcccc attctataca aaaacaacaa aaatgaaaag aaagttggac catggttctg
     1321 aggtccgctc tttttctttg ggaaagaaac catgcaaagt ctcagaatat acaagtacca
     1381 ctgggcttgt accatgttca gcaacaccaa caacttttgg ggacctcaga gcagccaatg
     1441 gccaagggca acaacgacgc cgaattacat ctgtccagcc acctacaggc ctccaggaat
     1501 ggctaaaaat gtttcagagc tggagtggac cagagaaatt gcttgcttta gatgaactca
     1561 ttgatagttg tgaaccaaca caagtaaaac atatgatgca agtgatagaa ccccagtttc
     1621 aacgagactt catttcattg ctccctaaag agttggcact ctatgtgctt tcattcctgg
     1681 aacccaaaga cctgctacaa gcagctcaga catgtcgcta ctggagaatt ttggctgaag
     1741 acaaccttct ctggagagag aaatgcaaag aagaggggat tgatgaacca ttgcacatca
     1801 agagaagaaa agtaataaaa ccaggtttca tacacagtcc atggaaaagt gcatacatca
     1861 gacagcacag aattgatact aactggaggc gaggagaact caaatctcct aaggtgctga
     1921 aaggacatga tgatcatgtg atcacatgct tacagttttg tggtaaccga atagttagtg
     1981 gttctgatga caacacttta agagtttggt cagcagtcac aggcaaatgt ctgagaacat
     2041 tagtgggaca tacaggtgga gtatggtcat cacaaatgag agacaacatc atcattagtg
     2101 gatctacaga tcggacactc aaagtgtgga atgcagagac tggagaatgt atacacacct
     2161 tatatgggca tacttccact gtgcgttgta tgcatcttca tgaaaaaaga gttgttagcg
     2221 gttctcgaga tgccactctt agggtttggg atattgagac aggccagtgt ttacatgttt
     2281 tgatgggtca tgttgcagca gtccgctgtg ttcaatatga tggcaggagg gttgttagtg
     2341 gagcatatga ttttatggta aaggtgtggg atccagagac tgaaacctgt ctacacacgt
     2401 tgcaggggca tactaataga gtctattcat tacagtttga tggtatccat gtggtgagtg
     2461 gatctcttga tacatcaatc cgtgtttggg atgtggagac agggaattgc attcacacgt
     2521 taacagggca ccagtcgtta acaagtggaa tggaactcaa agacaatatt cttgtctctg
     2581 ggaatgcaga ttctacagtt aaaatctggg atatcaaaac aggacagtgt ttacaaacat
     2641 tgcaaggtcc caacaagcat cagagtactg tgacctgttt acagttcaac aagaactttg
     2701 taattaccag ctcagatgat ggaactgtaa aactatggga cttgaaaacg ggtgaattta
     2761 ttcgaaacct agtcacattg gagagtgggg ggagtggggg agttgtgtgg cggatcagag
     2821 cctcaaacac aaagctggtg tgtgcagttg ggagtcggaa tgggactgaa gaaaccaagc
     2881 tgctggtgct ggactttgat gtggacatga agtgaaaccc agctttcttg tacaaagtgg
     2941 tctcgactct agaggatccc gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     3001 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     3061 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     3121 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     3181 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     3241 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     3301 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     3361 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     3421 ctcccttccc tgtccttctg attttaaaat aactatacca gcaggaggac gtccagacac
     3481 agcataggct acctggccat gcccaaccgg tgggacattt gagttgcttg cttggcactg
     3541 tcctctcatg cgttgggtcc actcagtaga tgcctgttga attgggtacg cggccagctt
     3601 ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3661 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3721 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3781 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3841 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3901 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcctc gaggaactga
     3961 aaaaccagaa agttaattcc ctatagtgag tcgtattaaa ttcgtaatca tgtcatagct
     4021 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4081 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4141 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4201 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4861 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4981 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5041 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5101 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5161 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5221 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5281 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5341 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5401 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5461 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5521 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5581 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5641 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5701 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5761 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5821 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5881 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5941 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6001 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6061 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6121 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg
     6181 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6241 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6301 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6361 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6421 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6481 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6541 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6601 aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     6661 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     6721 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc
     6781 aagctgatct atacattgaa tcaatattgg caattagcca tattagtcat tggttatata
     6841 gcataaatca at
//
