LOCUS       sequence-211588-        7866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miR-E (miR-30 variant)-based RNAi with GFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7866)
  AUTHORS   Fellmann C, Hoffmann T, Sridhar V, Hopfgartner B, Muhar M, Roth M, 
            Lai DY, Barbosa IA, Kwon JS, Guan Y, Sinha N, Zuber J
  TITLE     An optimized microRNA backbone for effective single-copy RNAi.
  JOURNAL   Cell Rep. 2013 Dec 26;5(6):1704-13. doi: 
            10.1016/j.celrep.2013.11.020. Epub 2013 Dec 12.
  PUBMED    24332856
REFERENCE   2  (bases 1 to 7866)
  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.celrep.2013.11.020"; journalName: "Cell Rep"; date: 
            "2013-12-26- 26"; volume: "5"; issue: "6"; pages: "1704-13"
COMMENT     Sequence Label: LEPG
FEATURES             Location/Qualifiers
     source          1..7866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     97..116
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             316..832
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    896..1237
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1304..1720
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1650..1672
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1688..1704
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1994..2493
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2029..2052)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2426..2445
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2514..3113
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2514..2533)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3010..3030
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3174..3741
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3191..3743
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3335..3352)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3562..3581
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3755..3757,3758..3760,3761..4474)
                     /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(3800..3821)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4061..4080)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4408..4429
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4576..5090
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5252..5268
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5276..5282,5283..5300,5301..5306))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5321..5342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5459..5476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5630..6218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5710..5729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6389..7180,7181..7249))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7012..7031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7250..7354)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7422..7440
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7478..7500)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7600..7619
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7813..7835
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
       61 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      121 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggcgatt
      181 agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga ctcaacaata
      241 tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt tatttagtct
      301 ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac
      361 gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta
      421 ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      481 ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag
      541 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      601 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      661 aaaagagccc acaacccctc actcggcgcg ccagtcctcc gatagactgc gtcgcccggg
      721 tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggac tcgctgatcc
      781 ttgggagggt ctcctcagat tgattgactg cccacctcgg gggtctttca tttggaggtt
      841 ccaccgagat ttggagaccc ctgcccaggg accaccgacc cccccgccgg gaggtaagct
      901 ggccagcggt cgtttcgtgt ctgtctctgt ctttgtgcgt gtttgtgccg gcatctaatg
      961 tttgcgcctg cgtctgtact agttagctaa ctagctctgt atctggcgga cccgtggtgg
     1021 aactgacgag ttctgaacac ccggccgcaa ccctgggaga cgtcccaggg actttggggg
     1081 ccgtttttgt ggcccgacct gaggaaggga gtcgatgtgg aatccgaccc cgtcaggata
     1141 tgtggttctg gtaggagacg agaacctaaa acagttcccg cctccgtctg aatttttgct
     1201 ttcggtttgg aaccgaagcc gcgcgtcttg tctgctgcag cgctgcagca tcgttctgtg
     1261 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac
     1321 tcccttaagt ttgaccttag gtcactggaa agatgtcgag cggatcgctc acaaccagtc
     1381 ggtagatgtc aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa
     1441 cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa
     1501 ggtcttttca cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga
     1561 agccttggct tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc
     1621 tcctcttcct ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg
     1681 atcctccctt tatccagccc tcactccttc tctaggcgcc ggaattagat ctctcgacta
     1741 gggataacag ggtaattgtt tgaatgaggc ttcagtactt tacagaatcg ttgcctgcac
     1801 atcttggaaa cacttgctgg gattacttcg acttcttaac ccaacagaag gctcgagcaa
     1861 ccagaattcg agcaattatc ttgtttacta aaactgaata ccttgctatc tctttgatac
     1921 atttttacaa agctgaatta aaatggtata aattaaatca cttttttcaa ttgacgcgtg
     1981 atctaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     2041 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     2101 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     2161 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     2221 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     2281 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     2341 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     2401 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     2461 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcccaagctt accatgaccg
     2521 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2581 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     2641 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2701 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     2761 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     2821 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     2881 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     2941 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3001 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3061 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     3121 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcc aattccgccc
     3181 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     3241 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     3301 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     3361 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     3421 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     3481 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     3541 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     3601 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     3661 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     3721 ctttgaaaaa cacgatgata atatggccac aaccatggtg agcaagggcg aggagctgtt
     3781 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3841 cgtgtctggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     3901 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     3961 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4021 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     4081 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     4141 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     4201 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg
     4261 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     4321 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     4381 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     4441 gatcactctc ggcatggacg agctgtacaa gtaatgaatt aattaagaat tatcaagctt
     4501 atcgataccg tcgacctgca gccaagctta tcgataaaat aaaagatttt atttagtctc
     4561 cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg
     4621 ccattttgca aggcatggaa aatacataac tgagaataga gaagttcaga tcaaggttag
     4681 gaacagagag acagcagaat atgggccaaa caggatatct gtggtaagca gttcctgccc
     4741 cggctcaggg ccaagaacag atggtcccca gatgcggtcc cgccctcagc agtttctaga
     4801 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
     4861 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
     4921 aaagagccca caacccctca ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt
     4981 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
     5041 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat gggtaacagt
     5101 ttcttgaagt tggagaacaa cattctgagg gtaggagtcg aatattaagt aatcctgact
     5161 caattagcca ctgttttgaa tccacatact ccaatactcc tgaaatagtt cattatggac
     5221 agcgcagaaa gagctgggga gaattgtgaa attgttatcc gctcacaatt ccacacaaca
     5281 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5461 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5521 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5581 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5641 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5701 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5761 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5821 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5881 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5941 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6001 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6061 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6121 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6181 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6241 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6301 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6361 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6421 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6481 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6541 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6601 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6661 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6721 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6781 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6841 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6901 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6961 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7021 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7081 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7141 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7201 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7261 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7321 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7381 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     7441 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     7501 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     7561 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     7621 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     7681 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     7741 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     7801 cgccagggtt ttcccagtca cgacgttgta aaacgacggc gcaaggaatg gtgcatgcaa
     7861 ggagat
//
