LOCUS       sequence-314281-        3300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3300)
  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 3300)
  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: pDONR221_eGFP (open)
FEATURES             Location/Qualifiers
     source          1..3300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     135..154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     388..405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(564..591)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(683..769)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     818..840
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     832..849
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     833..849
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    865..964
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(985..987,988..990,991..1701)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1030..1051)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1291..1310)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1638..1659
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1721..1820)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1837..1856)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1838..1856)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1861..1877)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1861..1877)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1979..1998)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1990..2799
                     /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
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2057..2076)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(2946..3300,1..234)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
       61 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      301 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      361 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      421 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      481 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgctagc
      541 caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt
      601 gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa
      661 cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
      721 agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag
      781 ttccctactc tcgcgttaac gctagcatgg atgttttccc agtcacgacg ttgtaaaacg
      841 acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat agtgacctgt
      901 tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta caaaaaagca
      961 ggctccgcgg ccgccccctt caccatggtg agcaagggcg aggagctgtt caccggggtg
     1021 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtctggc
     1081 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1141 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1201 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1261 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1321 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1381 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1441 atcatggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
     1501 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1561 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     1621 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     1681 ggcatggacg agctgtacaa gtccaagggt gggcgcgccg acccagcttt cttgtacaaa
     1741 gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt
     1801 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg
     1861 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     1921 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     1981 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     2041 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2101 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2161 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2221 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2281 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2341 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2401 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2461 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     2521 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     2581 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     2641 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     2701 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     2761 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     2821 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     2881 cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2941 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3001 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3061 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3121 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3181 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3241 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
//
