LOCUS       sequence-211468-        7676 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-regulated miR-E (miR-30 variant)-based RNAi with tGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7676)
  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 7676)
  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: RT3GEP
FEATURES             Location/Qualifiers
     source          1..7676
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        52..431
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        432..635
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     586..606
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             636..811
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    874..1073
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1274..1690
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1620..1642
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1658..1674
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1720..2098
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1728..1746
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1764..1782
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1800..1818
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1836..1854
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1872..1890
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1908..1926
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1944..1962
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2007..2031
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2119..2817
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     ncRNA           3008..3022
                     /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        3201..3700
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3236..3259)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3633..3652
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3721..4320
                     /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(3721..3740)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4217..4237
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4505..4930
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5033..5055)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5191..5211)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5196..5525
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5376..5511
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5547..5566
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5647..5664)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5818..6406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5898..5917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6577..7368,7369..7437))
                     /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     7200..7219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7438..7542)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7610..7628
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 atcataatat gtacatttat attggctcat gtccaacatt accgccatgt tgacattgat
       61 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      121 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      181 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      241 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      301 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      361 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      421 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      481 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      541 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      601 ggcgtgtacg gtgggaggtc tatataagca gagctcaata aaagagccca caacccctca
      661 ctcggcgcgc cagtcttccg atagactgcg tcgcccgggt acccgtattc ccaataaagc
      721 ctcttgctgt ttgcatccga atcgtggtct cgctgttcct tgggagggtc tcctctgagt
      781 gattgactac ccacgacggg ggtctttcat ttgggggctc gtccgggatt tggagacccc
      841 tgcccaggga ccaccgaccc accaccggga ggtaagctgg ccagcaactt atctgtgtct
      901 gtccgattgt ctagtgtcta tgtttgatgt tatgcgcctg cgtctgtact agttagctaa
      961 ctagctctgt atctggcgga cccgtggtgg aactgacgag ttctgaacac ccggccgcaa
     1021 ccctgggaga cgtcccaggg actttggggg ccgtttttgt ggcccgacct gaggaaggga
     1081 gtcgatgtgg aatccgaccc cgtcaggata tgtggttctg gtaggagacg agaacctaaa
     1141 acagttcccg cctccgtctg aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttg
     1201 tctgctgcag cgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt
     1261 gtctgaaaat tagggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa
     1321 agatgtcgag cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac
     1381 cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa
     1441 ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc
     1501 agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt
     1561 caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc
     1621 ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc
     1681 tctaggcgcc ggaattgaag atctatcgat tctagattcg agtttactcc ctatcagtga
     1741 tagagaacgt atgaagagtt tactccctat cagtgataga gaacgtatgc agactttact
     1801 ccctatcagt gatagagaac gtataaggag tttactccct atcagtgata gagaacgtat
     1861 gaccagttta ctccctatca gtgatagaga acgtatctac agtttactcc ctatcagtga
     1921 tagagaacgt atatccagtt tactccctat cagtgataga gaacgtataa gctttaggcg
     1981 tgtacggtgg gcgcctataa aagcagagct cgtttagtga accgtcagat cgcctggagc
     2041 aattccacaa cacttttgtc ttataccaac tttccgtacc acttcctacc ctcgtaaagt
     2101 cgagcttgcg ttggatccat ggagagcgac gagagcggcc tgcccgccat ggagatcgag
     2161 tgccgcatca ccggcaccct gaacggcgtg gagttcgagc tggtgggcgg cggagagggc
     2221 acccccgagc agggccgcat gaccaacaag atgaagagca ccaaaggcgc cctgaccttc
     2281 agcccctacc tgctgagcca cgtgatgggc tacggcttct accacttcgg cacctacccc
     2341 agcggctacg agaacccctt cctgcacgcc atcaacaacg gcggctacac caacacccgc
     2401 atcgagaagt acgaggacgg cggcgtgctg cacgtgagct tcagctaccg ctacgaggcc
     2461 ggccgcgtga tcggcgactt caaggtgatg ggcaccggct tccccgagga cagcgtgatc
     2521 ttcaccgaca agatcatccg cagcaacgcc accgtggagc acctgcaccc catgggcgat
     2581 aacgatctgg atggcagctt cacccgcacc ttcagcctgc gcgacggcgg ctactacagc
     2641 tccgtggtgg acagccacat gcacttcaag agcgccatcc accccagcat cctgcagaac
     2701 gggggcccca tgttcgcctt ccgccgcgtg gaggaggatc acagcaacac cgagctgggc
     2761 atcgtggagt accagcacgc cttcaagacc ccggatgcag atgccggtga agaataacgt
     2821 acggcggccg cctcgactag ggataacagg gtaattgttt gaatgaggct tcagtacttt
     2881 acagaatcgt tgcctgcaca tcttggaaac acttgctggg attacttcga cttcttaacc
     2941 caacagaagg ctcgagaagg tatattgctg ttgacagtga gcgcaggaat tataatgctt
     3001 atctatagtg aagccacaga tgtatagata agcattataa ttcctatgcc tactgcctcg
     3061 gacttcaagg ggctagaatt cgagcaatta tcttgtttac taaaactgaa taccttgcta
     3121 tctctttgat acatttttac aaagctgaat taaaatggta taaattaaat cacttttttc
     3181 aattgacgcg taattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     3241 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     3301 ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc
     3361 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     3421 gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca
     3481 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     3541 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
     3601 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     3661 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcagcc caagcttacc
     3721 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     3781 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     3841 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3901 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3961 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     4021 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     4081 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     4141 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     4201 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4261 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     4321 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcaggttcg
     4381 ataccgtcga gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga
     4441 agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc cagaaaaagg
     4501 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca
     4561 aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg
     4621 gaacagggtc gaccctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     4681 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     4741 ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga
     4801 ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     4861 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     4921 gtctttcatt tgggggctcg tccgggatcg ggagacccct gcccagggac caccgaccca
     4981 ccaccgggag gtaagctggc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     5041 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     5101 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     5161 gtagcgatag cggagtgtag atccggctgt ggaatgtgtg tcagttaggg tgtggaaagt
     5221 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5281 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5341 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5401 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5461 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5521 gcaaaaagct tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     5581 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     5641 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5701 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5761 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5821 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5881 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5941 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6001 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6061 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6121 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6181 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6241 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6301 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6361 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6421 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6481 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6541 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6601 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6661 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6721 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6781 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6841 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     6901 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6961 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7021 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7081 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7141 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     7201 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7261 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7321 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7381 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7441 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7501 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7561 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7621 cacgaggccc tttcgtcttc aagaattagc ttggccattg catacgttgt atccat
//
