LOCUS       sequence-314321-        4417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses murine Fbxw7 alpha with a T199 silent mutation in 
            mammalian cells. The T199 silent mutation confers resistance to the 
            5'-TGAACATGGTACAAGGCCAG-3' sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4417)
  AUTHORS   Gstalder C, Liu D, Miao D, Lutterbach B, DeVine AL, Lin C, Shettigar
            M, Pancholi P, Buchbinder EI, Carter SL, Manos MP, Rojas-Rudilla V, 
            Brennick R, Gjini E, Chen PH, Lako A, Rodig S, Yoon CH, Freeman GJ, 
            Barbie DA, Hodi FS, Miles W, Van Allen EM, Haq R
  TITLE     Inactivation of Fbxw7 impairs dsRNA sensing and confers resistance 
            to PD-1 blockade.
  JOURNAL   Cancer Discov. 2020 May 5. pii: 2159-8290.CD-19-1416. doi: 
            10.1158/2159-8290.CD-19-1416.
  PUBMED    32371478
REFERENCE   2  (bases 1 to 4417)
  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: "Cancer 
            Discov. 2020 May 5. pii: 2159-8290.CD-19-1416. doi: 
            10.1158/2159-8290.CD-19-1416."
COMMENT     Sequence Label: pENTR1A_mmFbxw7(res) (closed)
FEATURES             Location/Qualifiers
     source          1..4417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(2289..2388)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2500..2519)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2511..3320
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2578..2597)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3410..3998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3899..3918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4163..4249
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4341..4368
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     4376..4399
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct ttaaaggaac caattcagtc
      121 gactggatcc ggtaccgaat tcgcggccgc atgaatcagg aactgctctc tgtgggcagc
      181 aaaagacgac gaactggagg ctctctgaga gggaatgctt cctcaagcca ggttgatgag
      241 ggacagatga atcgcgtggt tgaggaggat ccacagcagc aagcgagaca tcaagaggag
      301 gagcacactg cgcggaatgg tgaacttgtg ggtgcaaacc ctaggcctgg agaccagaac
      361 gatacccagc aaggacaagt ggaggaaaat aataaccgct ttatttcagt agatgaggac
      421 tcttcgggaa atcaggaaga gcaagaggaa gatgaagagc atgctgggga acaggaggag
      481 gaagaggagg aagaggaaga ggaggaggag atggaccagg agagtgatga ttttgatccg
      541 tctgatgaca gtagcagaga agatgaacat acgcacaata gcaatgtcac aaactgcagt
      601 agtgtctcgg acctgcccgc tcaccagctc tcctctccat tctatacaaa gacaacaaaa
      661 atgaaaagaa agttggacca tggttctgaa gttcgttcct tttctttggg aaagaaacca
      721 tgcaaagtct cagattatac cagtacaact ggccttgtac catgttcagc aacaccaaca
      781 acttttgggg acctgagagc agccaatggg caagggcagc agcggcggag gattacatct
      841 gtccaaccac ccacaggcct tcaagagtgg ctgaaaatgt ttcagagctg gagcggacca
      901 gagaagttgc tggctttaga tgagctcatt gacagctgtg aaccaacaca agtgaagcat
      961 atgatgcaag tgatagagcc ccagttccag cgagacttca tctccttgct tcctaaagag
     1021 ttggcactct atgtgctttc attcctggaa cccaaagacc tgctgcaagc ggctcagact
     1081 tgtcgatact ggagaatttt ggctgaggat aaccttctct ggagagagaa atgtaaagaa
     1141 gaggggattg atgaaccgtt gcacatcaag agaagaaaaa taataaaacc aggtttcata
     1201 cacagcccat ggaagagtgc gtatatcaga cagcacagaa ttgatacaaa ctggagacga
     1261 ggagaactca aatctcctaa ggtgctgaaa gggcatgatg accatgtgat cacatgccta
     1321 cagttttgtg gcaaccgcat agttagtggt tctgatgaca acactttaaa agtttggtca
     1381 gcggtcacgg gcaagtgtct gagaacgtta gtgggacata caggtggagt gtggtcatca
     1441 cagatgagag acaatatcat catcagtgga tcgactgacc ggactctcaa agtgtggaat
     1501 gctgaaactg gagagtgtat acatacttta tatgggcaca cttctactgt acggtgtatg
     1561 catctccatg aaaaaagggt tgtaagcggt tctcgagatg ccactctcag ggtttgggat
     1621 attgagaccg gccagtgttt acacgtcctg atgggtcacg tagcagcggt ccgctgcgtt
     1681 cagtatgatg gcaggagggt tgttagtgga gcttatgatt ttatggtgaa ggtgtgggat
     1741 ccagagactg agacctgtct acacacgtta cagggacaca ctaatagagt ctattcatta
     1801 cagtttgatg gcatccatgt ggtgagtgga tctcttgata catcaatccg agtctgggat
     1861 gtggagacag ggaattgtat tcacacgcta acaggacacc agtcattaac gagtggaatg
     1921 gaactcaaag acaatattct tgtctctggg aatgcagatt ctacagttaa gatctgggat
     1981 atcaaaacag gacagtgttt acaaactttg caaggtccca gcaagcatca gagcgctgtg
     2041 acctgcttac agttcaacaa gaacttcgta attaccagct cagacgacgg aacggtcaaa
     2101 ctctgggact tgaaaacggg tgaatttatc cgaaacctcg tcacattgga gagtgggggg
     2161 agcgggggag ttgtgtggcg gatcagggcc tcaaacacaa agctggtgtg tgcagtcggg
     2221 agtcggaatg gaactgagga aaccaagctc ctggtgctgg actttgatgt ggacatgaaa
     2281 tgatctagac ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat
     2341 ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctgc
     2401 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     2461 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     2521 ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     2581 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga
     2641 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     2701 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     2761 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag
     2821 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     2881 tgtccctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     2941 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     3001 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     3061 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     3121 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     3181 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     3241 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     3301 tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacattat tcagattggg
     3361 ccccgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3421 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3481 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3541 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3601 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3661 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3721 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3781 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3841 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3901 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3961 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4021 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4081 attctgtgga taaccgtatt accgctagca tggatctcgg ggacgtctaa ctactaagcg
     4141 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcggaag actgggcctt
     4201 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     4261 ggatttgaac gttgtgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     4321 tgccaggcat caaactaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     4381 aactcttcct gttagttagt tacttaagct cgggccc
//
