LOCUS       sequence-209060-        7677 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 7677)
  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 7677)
  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..7677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     464..484
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             514..689
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    752..951
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1152..1568
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1498..1520
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1536..1552
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1598..1976
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1606..1624
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1642..1660
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1678..1696
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1714..1732
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1750..1768
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1786..1804
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1822..1840
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1885..1909
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1997..2695
                     /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           2886..2900
                     /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        3079..3578
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3114..3137)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3511..3530
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3599..4198
                     /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(3599..3618)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4095..4115
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4383..4808
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(4911..4933)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5069..5089)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5074..5403
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5254..5389
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5425..5444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5525..5542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5696..6284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5776..5795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6455..7246,7247..7315))
                     /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     7078..7097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7316..7420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7488..7506
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7606..7677,1..309)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgctctg gcattatgcc
      241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      301 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      361 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      421 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      481 cgtgtacggt gggaggtcta tataagcaga gctcaataaa agagcccaca acccctcact
      541 cggcgcgcca gtcttccgat agactgcgtc gcccgggtac ccgtattccc aataaagcct
      601 cttgctgttt gcatccgaat cgtggtctcg ctgttccttg ggagggtctc ctctgagtga
      661 ttgactaccc acgacggggg tctttcattt gggggctcgt ccgggatttg gagacccctg
      721 cccagggacc accgacccac caccgggagg taagctggcc agcaacttat ctgtgtctgt
      781 ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg tctgtactag ttagctaact
      841 agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc
      901 ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga ggaagggagt
      961 cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag aacctaaaac
     1021 agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc
     1081 tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt
     1141 ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt cactggaaag
     1201 atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct
     1261 tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc
     1321 gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag
     1381 accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca
     1441 agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc
     1501 ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc
     1561 taggcgccgg aattgaagat ctatcgattc tagattcgag tttactccct atcagtgata
     1621 gagaacgtat gaagagttta ctccctatca gtgatagaga acgtatgcag actttactcc
     1681 ctatcagtga tagagaacgt ataaggagtt tactccctat cagtgataga gaacgtatga
     1741 ccagtttact ccctatcagt gatagagaac gtatctacag tttactccct atcagtgata
     1801 gagaacgtat atccagttta ctccctatca gtgatagaga acgtataagc tttaggcgtg
     1861 tacggtgggc gcctataaaa gcagagctcg tttagtgaac cgtcagatcg cctggagcaa
     1921 ttccacaaca cttttgtctt ataccaactt tccgtaccac ttcctaccct cgtaaagtcg
     1981 agcttgcgtt ggatccatgg agagcgacga gagcggcctg cccgccatgg agatcgagtg
     2041 ccgcatcacc ggcaccctga acggcgtgga gttcgagctg gtgggcggcg gagagggcac
     2101 ccccgagcag ggccgcatga ccaacaagat gaagagcacc aaaggcgccc tgaccttcag
     2161 cccctacctg ctgagccacg tgatgggcta cggcttctac cacttcggca cctaccccag
     2221 cggctacgag aaccccttcc tgcacgccat caacaacggc ggctacacca acacccgcat
     2281 cgagaagtac gaggacggcg gcgtgctgca cgtgagcttc agctaccgct acgaggccgg
     2341 ccgcgtgatc ggcgacttca aggtgatggg caccggcttc cccgaggaca gcgtgatctt
     2401 caccgacaag atcatccgca gcaacgccac cgtggagcac ctgcacccca tgggcgataa
     2461 cgatctggat ggcagcttca cccgcacctt cagcctgcgc gacggcggct actacagctc
     2521 cgtggtggac agccacatgc acttcaagag cgccatccac cccagcatcc tgcagaacgg
     2581 gggccccatg ttcgccttcc gccgcgtgga ggaggatcac agcaacaccg agctgggcat
     2641 cgtggagtac cagcacgcct tcaagacccc ggatgcagat gccggtgaag aataacgtac
     2701 ggcggccgcc tcgactaggg ataacagggt aattgtttga atgaggcttc agtactttac
     2761 agaatcgttg cctgcacatc ttggaaacac ttgctgggat tacttcgact tcttaaccca
     2821 acagaaggct cgagaaggta tattgctgtt gacagtgagc gcaggaatta taatgcttat
     2881 ctatagtgaa gccacagatg tatagataag cattataatt cctatgccta ctgcctcgga
     2941 cttcaagggg ctagaattcg agcaattatc ttgtttacta aaactgaata ccttgctatc
     3001 tctttgatac atttttacaa agctgaatta aaatggtata aattaaatca cttttttcaa
     3061 ttgacgcgta attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg
     3121 ctttagcagc cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt
     3181 ccacatccac cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt
     3241 ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt
     3301 gacaaatgga agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat
     3361 ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg
     3421 ctcagaggct gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg
     3481 cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg
     3541 ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctgcagccca agcttaccat
     3601 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     3661 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     3721 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     3781 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     3841 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3901 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3961 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     4021 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     4081 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     4141 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg
     4201 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg caggttcgat
     4261 accgtcgaga tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag
     4321 cctatagagt acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg
     4381 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag
     4441 gcatggaaaa atacataact gagaatagag aagttcagat caaggtcagg aacagatgga
     4501 acagggtcga ccctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     4561 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     4621 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt
     4681 gactgagtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     4741 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     4801 ctttcatttg ggggctcgtc cgggatcggg agacccctgc ccagggacca ccgacccacc
     4861 accgggaggt aagctggctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4921 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4981 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     5041 agcgatagcg gagtgtagat ccggctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     5101 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     5161 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     5221 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     5281 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     5341 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     5401 aaaaagctta ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     5461 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     5521 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5581 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6121 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6421 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6481 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6541 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6601 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6661 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6721 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg
     6781 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6841 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6901 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6961 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7021 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     7081 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7141 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7201 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7261 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7321 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7381 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7441 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7501 cgaggccctt tcgtcttcaa gaattagctt ggccattgca tacgttgtat ccatatcata
     7561 atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga
     7621 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagt
//
