LOCUS       Exported                7845 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Tet-regulated miR-E (miR-30 variant)-based RNAi with tGFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    RT3GEN
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7845)
  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 7845)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7845
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             689..864
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    927..1126
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1327..1743
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1673..1695
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1711..1727
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1773..2151
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1781..1799
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1817..1835
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1853..1871
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1889..1907
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1925..1943
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1961..1979
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1997..2015
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2060..2084
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2172..2870
                     /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           3061..3075
                     /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        3254..3753
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3289..3312)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3686..3705
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3767..4570
                     /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(3830..3849)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4440..4459
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4727..5152
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5255..5277)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5413..5433)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5418..5747
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5598..5733
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5660..5679
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5769..5788
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5869..5886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6040..6628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6120..6139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6799..7659)
                     /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     7422..7441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7660..7764)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7832..5
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gaggcccttt cgtcttcaag aattagcttg gccattgcat acgttgtatc catatcataa
       61 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctca ataaaagagc ccacaacccc tcactcggcg
      721 cgccagtctt ccgatagact gcgtcgcccg ggtacccgta ttcccaataa agcctcttgc
      781 tgtttgcatc cgaatcgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac
      841 tacccacgac gggggtcttt catttggggg ctcgtccggg atttggagac ccctgcccag
      901 ggaccaccga cccaccaccg ggaggtaagc tggccagcaa cttatctgtg tctgtccgat
      961 tgtctagtgt ctatgtttga tgttatgcgc ctgcgtctgt actagttagc taactagctc
     1021 tgtatctggc ggacccgtgg tggaactgac gagttctgaa cacccggccg caaccctggg
     1081 agacgtccca gggactttgg gggccgtttt tgtggcccga cctgaggaag ggagtcgatg
     1141 tggaatccga ccccgtcagg atatgtggtt ctggtaggag acgagaacct aaaacagttc
     1201 ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa gccgcgcgtc ttgtctgctg
     1261 cagcgctgca gcatcgttct gtgttgtctc tgtctgactg tgtttctgta tttgtctgaa
     1321 aattagggcc agactgttac cactccctta agtttgacct taggtcactg gaaagatgtc
     1381 gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt taccttctgc
     1441 tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt taaccgagac
     1501 ctcatcaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca cccagaccag
     1561 gtcccctaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg ggtcaagccc
     1621 tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct cccccttgaa
     1681 cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc ttctctaggc
     1741 gccggaattg aagatctatc gattctagat tcgagtttac tccctatcag tgatagagaa
     1801 cgtatgaaga gtttactccc tatcagtgat agagaacgta tgcagacttt actccctatc
     1861 agtgatagag aacgtataag gagtttactc cctatcagtg atagagaacg tatgaccagt
     1921 ttactcccta tcagtgatag agaacgtatc tacagtttac tccctatcag tgatagagaa
     1981 cgtatatcca gtttactccc tatcagtgat agagaacgta taagctttag gcgtgtacgg
     2041 tgggcgccta taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg agcaattcca
     2101 caacactttt gtcttatacc aactttccgt accacttcct accctcgtaa agtcgagctt
     2161 gcgttggatc catggagagc gacgagagcg gcctgcccgc catggagatc gagtgccgca
     2221 tcaccggcac cctgaacggc gtggagttcg agctggtggg cggcggagag ggcacccccg
     2281 agcagggccg catgaccaac aagatgaaga gcaccaaagg cgccctgacc ttcagcccct
     2341 acctgctgag ccacgtgatg ggctacggct tctaccactt cggcacctac cccagcggct
     2401 acgagaaccc cttcctgcac gccatcaaca acggcggcta caccaacacc cgcatcgaga
     2461 agtacgagga cggcggcgtg ctgcacgtga gcttcagcta ccgctacgag gccggccgcg
     2521 tgatcggcga cttcaaggtg atgggcaccg gcttccccga ggacagcgtg atcttcaccg
     2581 acaagatcat ccgcagcaac gccaccgtgg agcacctgca ccccatgggc gataacgatc
     2641 tggatggcag cttcacccgc accttcagcc tgcgcgacgg cggctactac agctccgtgg
     2701 tggacagcca catgcacttc aagagcgcca tccaccccag catcctgcag aacgggggcc
     2761 ccatgttcgc cttccgccgc gtggaggagg atcacagcaa caccgagctg ggcatcgtgg
     2821 agtaccagca cgccttcaag accccggatg cagatgccgg tgaagaataa cgtacggcgg
     2881 ccgcctcgac tagggataac agggtaattg tttgaatgag gcttcagtac tttacagaat
     2941 cgttgcctgc acatcttgga aacacttgct gggattactt cgacttctta acccaacaga
     3001 aggctcgaga aggtatattg ctgttgacag tgagcgcagg aattataatg cttatctata
     3061 gtgaagccac agatgtatag ataagcatta taattcctat gcctactgcc tcggacttca
     3121 aggggctaga attcgagcaa ttatcttgtt tactaaaact gaataccttg ctatctcttt
     3181 gatacatttt tacaaagctg aattaaaatg gtataaatta aatcactttt ttcaattgac
     3241 gcgtaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     3301 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     3361 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
     3421 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
     3481 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
     3541 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
     3601 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
     3661 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
     3721 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gccaatatgg gatcggccat
     3781 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3841 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3901 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     3961 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4021 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4081 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4141 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4201 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4261 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4321 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4381 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4441 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4501 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4561 gttcttctga ggggatccgt cgacctgcag ccaagcttat cgataccgtc gagatatcag
     4621 tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc
     4681 catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     4741 cctgtaggtt tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaaatacat
     4801 aactgagaat agagaagttc agatcaaggt caggaacaga tggaacaggg tcgaccctag
     4861 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     4921 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     4981 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
     5041 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     5101 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
     5161 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
     5221 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     5281 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     5341 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     5401 agatccggct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     5461 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     5521 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     5581 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     5641 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     5701 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttactggct
     5761 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     5821 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     5881 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5941 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6001 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6061 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6121 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6181 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6241 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6301 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6361 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6421 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6481 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6541 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6601 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6661 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6721 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6781 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6841 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6901 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6961 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7021 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7081 agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc
     7141 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7201 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7261 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7321 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7381 tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag
     7441 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7501 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7561 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7621 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7681 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7741 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     7801 aaccattatt atcatgacat taacctataa aaataggcgt atcac
//
