LOCUS       sequence-99950-e         998 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  pENTR plasmid containing a mutated GFP followed by 3 miRNA haipins: 
            recommended as backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 998)
  AUTHORS   Myburgh R, Cherpin O, Schlaepfer E, Rehrauer H, Speck RF, Krause KH,
            Salmon P
  TITLE     Optimization of Critical Hairpin Features Allows miRNA-based Gene 
            Knockdown Upon Single-copy Transduction.
  JOURNAL   Mol Ther Nucleic Acids. 2014 Oct 28;3:e207. doi: 
            10.1038/mtna.2014.58.
  PUBMED    25350582
REFERENCE   2  (bases 1 to 998)
  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: "Mol Ther 
            Nucleic Acids."; date: "2014-10-28"; volume: "3:e207. doi"; pages: "
            10.1038/mtna.2014.58"
COMMENT     Sequence Label: pENTRdeltaGFPmirGE-DroshaT1.5.5
FEATURES             Location/Qualifiers
     source          1..998
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    7..106
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(134..136,137..850)
                     /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(134..136,137..847)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(176..197)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(437..456)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     784..805
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           936..950
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
ORIGIN
        1 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
       61 aagcaatgct tttttataat gccaactttg tacaaaaaag caggctccac catggatgga
      121 tgtaagtagg tgagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      181 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      241 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      301 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
      361 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      421 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      481 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      541 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
      601 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      661 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
      721 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
      781 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
      841 gtacaagtaa agcggcctga atcgccagtg tggatccatc gatactagtg gtgatagcaa
      901 tgtcagcagt gcctctcctc cacgaccaga ctttggtgaa gccacagatg caaagtctgg
      961 ccgtggaggg aaaagtaagg ttgaccatac tctactct
//
