LOCUS       sequence-99055-e        3349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pENTR plasmid containing a mutated GFP followed by 3 miRNA haipins: 
            recommended as backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3349)
  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 3349)
  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..3349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(485..487,488..1201)
                     /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(485..487,488..1198)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(527..548)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(788..807)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1135..1156
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1287..1301
                     /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"
     ncRNA           1400..1414
                     /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"
     ncRNA           1513..1527
                     /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"
     protein_bind    complement(1578..1677)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1789..1808)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1800..2609
                     /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
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1867..1886)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2699..3287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3188..3207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggctcca ccatggatgg atgtaagtag
      481 gtgagtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      541 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      601 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      661 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      721 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      781 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      841 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
      901 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
      961 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1021 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1081 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1141 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1201 aagcggcctg aatcgccagt gtggatccat cgatactagt ggtgatagca atgtcagcag
     1261 tgcctctcct ccacgaccag actttggtga agccacagat gcaaagtctg gccgtggagg
     1321 gaaaagtaag gttgaccata ctctactcta gtggtgatag caatgtcagc agtgcctctc
     1381 cctccacgac cagactttgg tgaagccaca gatgcaaagt ctggccgtgg agggaaaagt
     1441 aaggttgacc atactctact ctagtggtga tagcaatgtc agcagtgcct tattaaaact
     1501 gggaggtggg aagtgaagcc acagatgttc ccacctcaca gttttaatca agtaaggttg
     1561 accatactct actctagacc cagctttctt gtacaaagtt ggcattataa gaaagcattg
     1621 cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca
     1681 tccagctgca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     1741 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     1801 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     1861 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     1921 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     1981 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     2041 ccatcaagca ttttatccgt actcctggtg atgcatggtt actcaccact gcgatccccg
     2101 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     2161 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     2221 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     2281 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     2341 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     2401 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     2461 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     2521 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     2581 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacattatt
     2641 cagattgggc cccgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2701 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2761 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2821 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     2881 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2941 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3001 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3061 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3121 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3181 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3241 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3301 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtt
//
