LOCUS       sequence-211474-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-regulated miR-E (miR-30 variant)-based RNAi with DsRed reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  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 7827)
  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: RT3REN
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..27
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        507..710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     661..681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             711..886
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    949..1148
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1349..1765
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1695..1717
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1733..1749
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1802..2180
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1810..1828
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1846..1864
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1882..1900
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1918..1936
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1954..1972
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1990..2008
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2026..2044
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2089..2113
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2204..2881
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(2384..2404)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2796..2819
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           3065..3079
                     /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"
     promoter        3258..3757
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3293..3316)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3690..3709
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3771..4574
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3834..3853)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4444..4463
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4731..5156
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5259..5281)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5417..5437)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5422..5751
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5602..5737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5773..5792
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5873..5890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6044..6632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6124..6143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6803..7594,7595..7663))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7426..7445
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7664..7768)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattagct tggccattgc
       61 atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca acattaccgc
      121 catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct caataaaaga
      721 gcccacaacc cctcactcgg cgcgccagtc ttccgataga ctgcgtcgcc cgggtacccg
      781 tattcccaat aaagcctctt gctgtttgca tccgaatcgt ggtctcgctg ttccttggga
      841 gggtctcctc tgagtgattg actacccacg acgggggtct ttcatttggg ggctcgtccg
      901 ggatttggag acccctgccc agggaccacc gacccaccac cgggaggtaa gctggccagc
      961 aacttatctg tgtctgtccg attgtctagt gtctatgttt gatgttatgc gcctgcgtct
     1021 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
     1081 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
     1141 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
     1201 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
     1261 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
     1321 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1381 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1441 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1501 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1561 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1621 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1681 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1741 agccctcact ccttctctag gcgccggaat tgaagatctg ggggatcgat cctctagatt
     1801 cgagtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     1861 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     1921 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     1981 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     2041 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     2101 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     2161 ccacttccta ccctcgtaaa gtcgagcttg cgttggatcc accatggcct cctccgagaa
     2221 cgtcatcacc gagttcatgc gcttcaaggt gcgcatggag ggcaccgtga acggccacga
     2281 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggccacaaca ccgtgaagct
     2341 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca
     2401 gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc
     2461 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggcgac
     2521 cgtgacccag gactcctccc tgcaggacgg ctgcttcatc tacaaggtga agttcatcgg
     2581 cgtgaacttc ccctccgacg gccccgtgat gcagaagaag accatgggct gggaggcctc
     2641 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagacccaca aggccctgaa
     2701 gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc
     2761 cgtgcagctg cccggctact actacgtgga cgccaagctg gacatcacct cccacaacga
     2821 ggactacacc atcgtggagc agtacgagcg caccgagggc cgccaccacc tgttcctgta
     2881 gcggccgcct cgactaggga taacagggta attgtttgaa tgaggcttca gtactttaca
     2941 gaatcgttgc ctgcacatct tggaaacact tgctgggatt acttcgactt cttaacccaa
     3001 cagaaggctc gagaaggtat attgctgttg acagtgagcg caggaattat aatgcttatc
     3061 tatagtgaag ccacagatgt atagataagc attataattc ctatgcctac tgcctcggac
     3121 ttcaaggggc tagaattcga gcaattatct tgtttactaa aactgaatac cttgctatct
     3181 ctttgataca tttttacaaa gctgaattaa aatggtataa attaaatcac ttttttcaat
     3241 tgacgcgtaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     3301 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     3361 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     3421 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     3481 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     3541 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     3601 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     3661 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     3721 cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcagccaat atgggatcgg
     3781 ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3841 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3901 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3961 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4021 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4081 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4141 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4201 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4261 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4321 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4381 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4441 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4501 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4561 acgagttctt ctgaggggat ccgtcgacct gcagccaagc ttatcgatac cgtcgagata
     4621 tcagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac
     4681 gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     4741 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat
     4801 acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac agggtcgacc
     4861 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta
     4921 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
     4981 caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc
     5041 cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt
     5101 tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatttggg
     5161 ggctcgtccg ggatcgggag acccctgccc agggaccacc gacccaccac cgggaggtaa
     5221 gctggctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     5281 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     5341 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     5401 gtgtagatcc ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     5461 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     5521 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     5581 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5641 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5701 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttact
     5761 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     5821 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     5881 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5941 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6001 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6061 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6121 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6181 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6241 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6301 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6361 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6421 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6481 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6541 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6601 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6661 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6721 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6781 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6841 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6901 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6961 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7021 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7081 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     7141 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7201 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7261 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7321 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7381 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     7441 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7501 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7561 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7621 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7681 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7741 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     7801 aagaaaccat tattatcatg acattaa
//
