LOCUS       sequence-394874-        8466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid expresses GST-tagged FBW7 human isoform 1. Used for in 
            vitro translation of FBW7alpha..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8466)
  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 8466)
  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-GST-FBW7 alpha
FEATURES             Location/Qualifiers
     source          1..8466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             123..770
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="PILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFE
                     LGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV
                     SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYM
                     DPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     728..750
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             777..794
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    801..825
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2950..2974)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3034..3066
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3073..3090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3256..3342
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3434..3461
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3515..3533)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3529..3576
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3613..4068
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3700..3719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3910..3931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4183..4974,4975..5043))
                     /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     4806..4825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5044..5136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5217..5805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5706..5725
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5959..5976
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5991..6130
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6056..6075)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6216..6238
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6232..6423)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6995..8077)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8039..8058
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8078..8155)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8361..8380
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatatacata tgagatccca tcaccatcac catcacggcg
      121 cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg
      181 aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc
      241 gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg
      301 atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt
      361 tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata
      421 ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt
      481 ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat
      541 atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg
      601 ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac
      661 gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc
      721 ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg
      781 ttccgcgtgg atctgatatc acaagtttgt acaaaaaagc aggctttaat caggaactgc
      841 tctctgtggg cagcaaaaga cgacgaactg gaggctctct gagaggtaac ccttcctcaa
      901 gccaggtaga tgaagaacag atgaatcgtg tggtagagga ggaacagcaa cagcaactca
      961 gacaacaaga ggaggagcac actgcaagga atggtgaagt tgttggagta gaacctagac
     1021 ctggaggcca aaatgattcc cagcaaggac agttggaaga aaacaataat agatttattt
     1081 cggtagatga ggactcctca ggaaaccaag aagaacaaga ggaagatgaa gaacatgctg
     1141 gtgaacaaga tgaggaggat gaggaggagg aggagatgga ccaggagagt gacgattttg
     1201 atcagtctga tgatagtagc agagaagatg aacatacaca tactaacagt gtcacgaact
     1261 ccagtagtat tgtggacctg cccgttcacc aactctcctc cccattctat acaaaaacaa
     1321 caaaaatgaa aagaaagttg gaccatggtt ctgaggtccg ctctttttct ttgggaaaga
     1381 aaccatgcaa agtctcagaa tatacaagta ccactgggct tgtaccatgt tcagcaacac
     1441 caacaacttt tggggacctc agagcagcca atggccaagg gcaacaacga cgccgaatta
     1501 catctgtcca gccacctaca ggcctccagg aatggctaaa aatgtttcag agctggagtg
     1561 gaccagagaa attgcttgct ttagatgaac tcattgatag ttgtgaacca acacaagtaa
     1621 aacatatgat gcaagtgata gaaccccagt ttcaacgaga cttcatttca ttgctcccta
     1681 aagagttggc actctatgtg ctttcattcc tggaacccaa agacctgcta caagcagctc
     1741 agacatgtcg ctactggaga attttggctg aagacaacct tctctggaga gagaaatgca
     1801 aagaagaggg gattgatgaa ccattgcaca tcaagagaag aaaagtaata aaaccaggtt
     1861 tcatacacag tccatggaaa agtgcataca tcagacagca cagaattgat actaactgga
     1921 ggcgaggaga actcaaatct cctaaggtgc tgaaaggaca tgatgatcat gtgatcacat
     1981 gcttacagtt ttgtggtaac cgaatagtta gtggttctga tgacaacact ttaaaagttt
     2041 ggtcagcagt cacaggcaaa tgtctgagaa cattagtggg acatacaggt ggagtatggt
     2101 catcacaaat gagagacaac atcatcatta gtggatctac agatcggaca ctcaaagtgt
     2161 ggaatgcaga gactggagaa tgtatacaca ccttatatgg gcatacttcc actgtgcgtt
     2221 gtatgcatct tcatgaaaaa agagttgtta gcggttctcg agatgccact cttagggttt
     2281 gggatattga gacaggccag tgtttacatg ttttgatggg tcatgttgca gcagtccgct
     2341 gtgttcaata tgatggcagg agggttgtta gtggagcata tgattttatg gtaaaggtgt
     2401 gggatccaga gactgaaacc tgtctacaca cgttgcaggg gcatactaat agagtctatt
     2461 cattacagtt tgatggtatc catgtggtga gtggatctct tgatacatca atccgtgttt
     2521 gggatgtgga gacagggaat tgcattcaca cgttaacagg gcaccagtcg ttaacaagtg
     2581 gaatggaact caaagacaat attcttgtct ctgggaatgc agattctaca gttaaaatct
     2641 gggatatcaa aacaggacag tgtttacaaa cattgcaagg tcccaacaag catcagagta
     2701 ctgtgacctg tttacagttc aacaagaact ttgtaattac cagctcagat gatggaactg
     2761 taaaactatg ggacttgaaa acgggtgaat ttattcgaaa cctagtcaca ttggagagtg
     2821 gggggagtgg gggagttgtg tggcggatca gagcctcaaa cacaaagctg gtgtgtgcag
     2881 ttgggagtcg gaatgggact gaagaaacca agctgctggt gctggacttt gatgtggaca
     2941 tgaagtgaaa cccagctttc ttgtacaaag tggtgatatc gctagctaga tagctagaag
     3001 cttgcggccg cacagctgta tacacgtgca agccagccag aactcgctcc tgaagaccca
     3061 gaggatctcg agcaccacca ccaccaccac taatgttaat taagttgggc gttcctaggc
     3121 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     3181 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     3241 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3301 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     3361 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     3421 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     3481 ttgtttattt ttctaaatac attcaaatat gtatccgctg agcaataact agcataaccc
     3541 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     3601 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3661 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3721 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3781 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3841 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3901 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3961 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4021 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttct ggcggcacga
     4081 tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4141 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4201 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4261 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4321 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4381 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4441 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4501 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4561 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4621 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4681 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4741 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4801 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4861 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4921 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4981 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5041 catactcttc ctttttcaat catgattgaa gcatttatca gggttattgt ctcatgagcg
     5101 gatacatatt tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa aatcccttaa
     5161 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5221 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5281 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5341 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5401 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5461 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5521 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5581 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5641 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5701 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5761 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5821 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5881 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5941 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     6001 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     6061 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     6121 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6181 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6241 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     6301 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     6361 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     6421 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     6481 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     6541 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     6601 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     6661 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     6721 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     6781 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     6841 ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca
     6901 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     6961 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     7021 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     7081 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     7141 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     7201 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     7261 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     7321 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     7381 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     7441 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     7501 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     7561 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     7621 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     7681 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     7741 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     7801 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     7861 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     7921 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     7981 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     8041 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     8101 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     8161 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     8221 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     8281 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     8341 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     8401 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     8461 gcgaaa
//
