LOCUS       sequence-486040-        3336 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Middle entry fluorescent GFP reporter with a viral 2A peptide for 
            use with the Gateway cloning system; allows 3' transgene to be 
            transcribed on the same mRNA but translated separately.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3336)
  AUTHORS   Kendall GC, Watson S, Xu L, LaVigne CA, Murchison W, Rakheja D, 
            Skapek SX, Tirode F, Delattre O, Amatruda JF
  TITLE     PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator 
            of rhabdomyosarcoma tumorigenesis.
  JOURNAL   eLife. 2018 Jun 5;7. pii: 33800. doi: 10.7554/eLife.33800.
  PUBMED    29869612
REFERENCE   2  (bases 1 to 3336)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eLife."; 
            date: "2018-06-5"; volume: "7. pii: 33800. doi"; pages: " 
            10.7554/eLife.33800"
COMMENT     Sequence Label: mE-GFP2A
FEATURES             Location/Qualifiers
     source          1..3336
                     /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"
     regulatory      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(722..743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(983..1002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1330..1351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1403..1459
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(1461..1560)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1577..1596)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1578..1596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1601..1617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1601..1617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1719..1738)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1730..2539
                     /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(1797..1816)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2686..3274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3175..3194
                     /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 agcaggctgc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1381 cgagctgtac aagggatccg gagccacgaa cttctctctg ttaaagcaag caggagacgt
     1441 ggaagaaaac cccggtcctg acccagcttt cttgtacaaa gttggcatta taagaaagca
     1501 ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg
     1561 ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca
     1621 gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     1681 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     1741 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     1801 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa
     1861 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     1921 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     1981 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc
     2041 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     2101 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     2161 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     2221 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     2281 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     2341 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     2401 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     2461 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     2521 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     2581 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc
     2641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2821 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3301 gttcctggcc ttttgctggc cttttgctca catgtt
//
