LOCUS       sequence-413683-        3510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible entry vector, with insert of the EGFP gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3510)
  AUTHORS   Xiao MS, Damodaran AP, Kumari B, Dickson E, Xing K, On TA, Parab N, 
            King HE, Perez AR, Guiblet WM, Duncan G, Che A, Chari R, Andresson 
            T, Vidigal JA, Weatheritt RJ, Aregger M, Gonatopoulos-Pournatzis T
  TITLE     Genome-scale exon perturbation screens uncover exons critical for 
            cell fitness.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2553-2572.e19. doi: 
            10.1016/j.molcel.2024.05.024. Epub 2024 Jun 24.
  PUBMED    38917794
REFERENCE   2  (bases 1 to 3510)
  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"; doi: 
            "10.1016/j.molcel.2024.05.024"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2553-2572.e19"
COMMENT     Sequence Label: pDONR223-EGFP
FEATURES             Location/Qualifiers
     source          1..3510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(671..673,674..1387)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(671..673,674..1384)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(713..734)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(974..993)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1321..1342
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1389..1488)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1505..1524)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1506..1524)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1529..1545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1529..1545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1976..2767
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2860..3448
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3349..3368
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggcttc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
      721 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
      781 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
      841 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
      901 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
      961 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1021 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1081 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1141 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1201 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1261 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1321 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1381 caagtaaaac ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat
     1441 ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga
     1501 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
     1561 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gcctcctcta
     1621 gaccagccag gacagaaatg cctcgacttc gctgctaccc aaggttgccg ggtgacgcac
     1681 accgtggaaa cggatgaagg cacgaaccca gtggacataa gcctgttcgg ttcgtaagct
     1741 gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg
     1801 tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc
     1861 tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg
     1921 gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa acattatgag
     1981 ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg
     2041 ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct
     2101 gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac
     2161 gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat
     2221 tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc
     2281 agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt
     2341 cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag
     2401 cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct
     2461 atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga
     2521 tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat
     2581 cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc
     2641 cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg
     2701 cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg
     2761 caaataaccc tcgagccacc catgaccaaa atcccttaac gtgagttacg cgtcgttcca
     2821 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2881 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2941 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3001 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3061 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3121 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3181 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3241 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3301 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3361 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3421 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3481 ggccttttgc tggccttttg ctcacatgtt
//
