LOCUS       sequence-209061-        7846 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 7846)
  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 7846)
  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: RT3GEN
FEATURES             Location/Qualifiers
     source          1..7846
                     /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             3592..4395
                     /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(3655..3674)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4265..4284
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4552..4977
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5080..5102)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5238..5258)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5243..5572
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5423..5558
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5594..5613
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5694..5711)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5865..6453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5945..5964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6624..7415,7416..7484))
                     /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     7247..7266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7485..7589)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7657..7675
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7775..7846,1..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 ctgcagccaa tatgggatcg
     3601 gccattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3661 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3721 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3781 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3841 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3901 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3961 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4021 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4081 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4141 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4201 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4261 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4321 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4381 gacgagttct tctgagggga tccgtcgacc tgcagccaag cttatcgata ccgtcgagat
     4441 atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc ctatagagta
     4501 cgagccatag ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     4561 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa
     4621 tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa cagggtcgac
     4681 cctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
     4741 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
     4801 tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg actgagtcgc
     4861 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
     4921 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatttgg
     4981 gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca ccgggaggta
     5041 agctggctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5101 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     5161 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     5221 agtgtagatc cggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     5281 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     5341 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     5401 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     5461 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     5521 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagcttac
     5581 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     5641 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     5701 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5761 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5821 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5881 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5941 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6001 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6061 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6121 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6181 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6241 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6301 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6361 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6421 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6481 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6541 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6601 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6661 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6721 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6781 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6841 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6901 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     6961 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7021 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7081 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7141 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7201 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     7261 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7321 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7381 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7441 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7501 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7561 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7621 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     7681 cgtcttcaag aattagcttg gccattgcat acgttgtatc catatcataa tatgtacatt
     7741 tatattggct catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa
     7801 tagtaatcaa ttacggggtc attagttcat agcccatata tggagt
//
