LOCUS       sequence-209062-        7828 bp    DNA     circular SYN 14-APR-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 7828)
  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 7828)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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..7828
                     /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        1605..1983
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1613..1631
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1649..1667
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1685..1703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1721..1739
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1757..1775
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1793..1811
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1829..1847
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1892..1916
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2007..2684
                     /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(2187..2207)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2599..2622
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           2868..2882
                     /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        3061..3560
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3096..3119)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3493..3512
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3574..4377
                     /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(3637..3656)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4247..4266
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4534..4959
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5062..5084)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5220..5240)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5225..5554
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5405..5540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5576..5595
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5676..5693)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5847..6435)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5927..5946)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6606..7397,7398..7466))
                     /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     7229..7248
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7467..7571)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7639..7657
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7757..7828,1..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 ctgggggatc gatcctctag attcgagttt actccctatc
     1621 agtgatagag aacgtatgaa gagtttactc cctatcagtg atagagaacg tatgcagact
     1681 ttactcccta tcagtgatag agaacgtata aggagtttac tccctatcag tgatagagaa
     1741 cgtatgacca gtttactccc tatcagtgat agagaacgta tctacagttt actccctatc
     1801 agtgatagag aacgtatatc cagtttactc cctatcagtg atagagaacg tataagcttt
     1861 aggcgtgtac ggtgggcgcc tataaaagca gagctcgttt agtgaaccgt cagatcgcct
     1921 ggagcaattc cacaacactt ttgtcttata ccaactttcc gtaccacttc ctaccctcgt
     1981 aaagtcgagc ttgcgttgga tccaccatgg cctcctccga gaacgtcatc accgagttca
     2041 tgcgcttcaa ggtgcgcatg gagggcaccg tgaacggcca cgagttcgag atcgagggcg
     2101 agggcgaggg ccgcccctac gagggccaca acaccgtgaa gctgaaggtg accaagggcg
     2161 gccccctgcc cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt
     2221 acgtgaagca ccccgccgac atccccgact acaagaagct gtccttcccc gagggcttca
     2281 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggc gaccgtgacc caggactcct
     2341 ccctgcagga cggctgcttc atctacaagg tgaagttcat cggcgtgaac ttcccctccg
     2401 acggccccgt gatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc
     2461 cccgcgacgg cgtgctgaag ggcgagaccc acaaggccct gaagctgaag gacggcggcc
     2521 actacctggt ggagttcaag tccatctaca tggccaagaa gcccgtgcag ctgcccggct
     2581 actactacgt ggacgccaag ctggacatca cctcccacaa cgaggactac accatcgtgg
     2641 agcagtacga gcgcaccgag ggccgccacc acctgttcct gtagcggccg cctcgactag
     2701 ggataacagg gtaattgttt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca
     2761 tcttggaaac acttgctggg attacttcga cttcttaacc caacagaagg ctcgagaagg
     2821 tatattgctg ttgacagtga gcgcaggaat tataatgctt atctatagtg aagccacaga
     2881 tgtatagata agcattataa ttcctatgcc tactgcctcg gacttcaagg ggctagaatt
     2941 cgagcaatta tcttgtttac taaaactgaa taccttgcta tctctttgat acatttttac
     3001 aaagctgaat taaaatggta taaattaaat cacttttttc aattgacgcg taattctacc
     3061 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     3121 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     3181 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     3241 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     3301 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     3361 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     3421 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     3481 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     3541 cctcatctcc gggcctttcg acctgcagcc aatatgggat cggccattga acaagatgga
     3601 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3661 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3721 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3781 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3841 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3901 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3961 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4021 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4081 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4141 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4201 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4261 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4321 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaggg
     4381 gatccgtcga cctgcagcca agcttatcga taccgtcgag atatcagtgg tccaggctct
     4441 agttttgact caacaatatc accagctgaa gcctatagag tacgagccat agataaaata
     4501 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     4561 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     4621 gaagttcaga tcaaggtcag gaacagatgg aacagggtcg accctagaga accatcagat
     4681 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
     4741 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
     4801 acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc
     4861 aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc
     4921 tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt ccgggatcgg
     4981 gagacccctg cccagggacc accgacccac caccgggagg taagctggct gcctcgcgcg
     5041 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     5101 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     5161 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtaga tccggctgtg
     5221 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5281 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     5341 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     5401 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5461 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     5521 aggaggcttt tttggaggcc taggcttttg caaaaagctt actggcttaa ctatgcggca
     5581 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     5641 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5701 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5761 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5821 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5881 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5941 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6001 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6061 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6121 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6181 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6241 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     6301 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6361 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6421 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6481 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6541 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6601 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6661 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6721 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6781 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6841 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6901 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6961 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7021 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7081 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7141 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7201 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     7261 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7321 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7381 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7441 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7501 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7561 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7621 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattagct
     7681 tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca
     7741 acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     7801 tcattagttc atagcccata tatggagt
//
