LOCUS       sequence-260922-        8077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miR-E (miR-30 variant)-based RNAi with mCherry reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8077)
  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 8077)
  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: LENC
FEATURES             Location/Qualifiers
     source          1..8077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     155..174
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             374..890
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    954..1295
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1362..1778
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1708..1730
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1746..1762
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1967..1981
                     /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        2165..2664
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2200..2223)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2597..2616
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2678..3481
                     /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(2741..2760)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3351..3370
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3487..4054
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3504..4056
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3648..3665)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3875..3894
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4068..4778
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4213..4231)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4481..4500
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             4845..5359
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5521..5537
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5545..5551,5552..5569,5570..5575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5590..5611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5728..5745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5899..6487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5979..5998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6658..7449,7450..7518))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7281..7300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7519..7623)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7691..7709
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7747..7769)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7869..7888
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg
       61 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      121 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      181 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggcgattag
      241 tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc
      301 accagctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
      361 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
      421 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
      481 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
      541 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
      601 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
      661 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
      721 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
      781 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt
      841 gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc
      901 accgagattt ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg
      961 ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt
     1021 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1081 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1141 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1201 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1261 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1321 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     1381 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1441 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1501 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1561 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1621 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1681 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1741 cctcccttta tccagccctc actccttctc taggcgccgg aattagatct ctcgactagg
     1801 gataacaggg taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat
     1861 cttggaaaca cttgctggga ttacttcgac ttcttaaccc aacagaaggc tcgagaaggt
     1921 atattgctgt tgacagtgag cgcaggaatt ataatgctta tctatagtga agccacagat
     1981 gtatagataa gcattataat tcctatgcct actgcctcgg acttcaaggg gctagaattc
     2041 gagcaattat cttgtttact aaaactgaat accttgctat ctctttgata catttttaca
     2101 aagctgaatt aaaatggtat aaattaaatc acttttttca attgacgcgt gatctaattc
     2161 taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     2221 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     2281 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     2341 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     2401 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     2461 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     2521 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     2581 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     2641 tcttcctcat ctccgggcct ttcgacctgc agccaatatg ggatcggcca ttgaacaaga
     2701 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     2761 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     2821 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     2881 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     2941 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3001 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3061 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3121 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3181 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     3241 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3301 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3361 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3421 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3481 agcaattccg cccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     3541 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     3601 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     3661 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     3721 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     3781 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     3841 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     3901 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     3961 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     4021 ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gtgagcaagg
     4081 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     4141 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     4201 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     4261 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     4321 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     4381 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     4441 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     4501 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     4561 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     4621 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     4681 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     4741 gccactccac cggcggcatg gacgagctgt acaagtaagt cgacctgcag ccaagcttat
     4801 cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     4861 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact
     4921 gagaatagag aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac
     4981 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag
     5041 atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag
     5101 gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt
     5161 tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc
     5221 agtcctccga tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt
     5281 gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc
     5341 gtcagcgggg gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg
     5401 taggagtcga atattaagta atcctgactc aattagccac tgttttgaat ccacatactc
     5461 caatactcct gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa
     5521 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5581 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5641 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5701 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5761 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5821 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5881 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5941 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6001 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6061 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6121 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6181 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6241 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6301 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6361 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6421 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6481 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6541 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6601 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6661 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6721 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6781 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6841 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6901 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6961 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7021 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7081 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7141 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7201 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7261 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7321 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7381 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7441 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7501 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7561 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7621 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     7681 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     7741 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     7801 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     7861 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     7921 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     7981 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     8041 atgtgctgca aggcgattaa gttgggtaac gccaggg
//
