LOCUS       sequence-211490-        7905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miR-E (miR-30 variant)-based RNAi with ZsGreen reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7905)
  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 7905)
  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: LEPZ
FEATURES             Location/Qualifiers
     source          1..7905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             175..691
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    755..1096
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1163..1579
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1509..1531
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1547..1563
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1768..1782
                     /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        1966..2465
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2001..2024)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2398..2417
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2486..3085
                     /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(2486..2505)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2982..3002
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3146..3713
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3163..3715
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3307..3324)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3534..3553
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3727..4422
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     LTR             4474..4988
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5150..5166
                     /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(5174..5180,5181..5198,5199..5204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5219..5240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5357..5374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5528..6116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5608..5627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6287..7078,7079..7147))
                     /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     6910..6929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7148..7252)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7320..7338
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7376..7398)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7498..7517
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7711..7733
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7861..7880
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggtgatgccg gccacgatgc gtccggcgta gaggcgatta gtccaatttg ttaaagacag
       61 gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga
      121 gtacgagcca tagataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
      181 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
      241 aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat
      301 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
      361 atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag
      421 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
      481 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
      541 ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtattc ccaataaagc
      601 ctcttgctgt ttgcatccga atcgtggact cgctgatcct tgggagggtc tcctcagatt
      661 gattgactgc ccacctcggg ggtctttcat ttggaggttc caccgagatt tggagacccc
      721 tgcccaggga ccaccgaccc ccccgccggg aggtaagctg gccagcggtc gtttcgtgtc
      781 tgtctctgtc tttgtgcgtg tttgtgccgg catctaatgt ttgcgcctgc gtctgtacta
      841 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tctgaacacc
      901 cggccgcaac cctgggagac gtcccaggga ctttgggggc cgtttttgtg gcccgacctg
      961 aggaagggag tcgatgtgga atccgacccc gtcaggatat gtggttctgg taggagacga
     1021 gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttgga accgaagccg
     1081 cgcgtcttgt ctgctgcagc gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt
     1141 tctgtatttg tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg
     1201 tcactggaaa gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg
     1261 ttgggttacc ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg
     1321 cacctttaac cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca
     1381 tggacaccca gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc
     1441 tccctgggtc aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc
     1501 gtctctcccc cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct
     1561 cactccttct ctaggcgccg gaattagatc tctcgactag ggataacagg gtaattgttt
     1621 gaatgaggct tcagtacttt acagaatcgt tgcctgcaca tcttggaaac acttgctggg
     1681 attacttcga cttcttaacc caacagaagg ctcgagaagg tatattgctg ttgacagtga
     1741 gcgcaggaat tataatgctt atctatagtg aagccacaga tgtatagata agcattataa
     1801 ttcctatgcc tactgcctcg gacttcaagg ggctagaatt cgagcaatta tcttgtttac
     1861 taaaactgaa taccttgcta tctctttgat acatttttac aaagctgaat taaaatggta
     1921 taaattaaat cacttttttc aattgacgcg tgatctaatt ctaccgggta ggggaggcgc
     1981 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     2041 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     2101 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     2161 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     2221 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     2281 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     2341 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     2401 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     2461 tttcgacctg cagcccaagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     2521 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     2581 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     2641 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     2701 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     2761 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     2821 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     2881 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     2941 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3001 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3061 catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     3121 gagcgcacga ccccatgcat ccaattccgc cccccccccc taacgttact ggccgaagcc
     3181 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt
     3241 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc
     3301 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc
     3361 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc
     3421 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg
     3481 cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct
     3541 cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat
     3601 ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc
     3661 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc
     3721 acaaccatgg cccagtccaa gcacggcctg accaaggaga tgaccatgaa gtaccgcatg
     3781 gagggctgcg tggacggcca caagttcgtg atcaccggcg agggcatcgg ctaccccttc
     3841 aagggcaagc aggccatcaa cctgtgcgtg gtggagggcg gccccttgcc cttcgccgag
     3901 gacatcttgt ccgccgcctt catgtacggc aaccgcgtgt tcaccgagta cccccaggac
     3961 atcgtcgact acttcaagaa ctcctgcccc gccggctaca cctgggaccg ctccttcctg
     4021 ttcgaggacg gcgccgtgtg catctgcaac gccgacatca ccgtgagcgt ggaggagaac
     4081 tgcatgtacc acgagtccaa gttctacggc gtgaacttcc ccgccgacgg ccccgtgatg
     4141 aagaagatga ccgacaactg ggagccctcc tgcgagaaga tcatccccgt gcccaagcag
     4201 ggcatcttga agggcgacgt gagcatgtac ctgctgctga aggacggtgg ccgcttgcgc
     4261 tgccagttcg acaccgtgta caaggccaag tccgtgcccc gcaagatgcc cgactggcac
     4321 ttcatccagc acaagctgac ccgcgaggac cgcagcgacg ccaagaacca gaagtggcac
     4381 ctgaccgagc acgccatcgc ctccggctcc gccttgccct gaagatcatc gataaaataa
     4441 aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc
     4501 aagctagctt aagtaacgcc attttgcaag gcatggaaaa tacataactg agaatagaga
     4561 agttcagatc aaggttagga acagagagac agcagaatat gggccaaaca ggatatctgt
     4621 ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtcccg
     4681 ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg
     4741 accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc
     4801 tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca gtcctccgat
     4861 agactgcgtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt
     4921 gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg
     4981 tctttcatgg gtaacagttt cttgaagttg gagaacaaca ttctgagggt aggagtcgaa
     5041 tattaagtaa tcctgactca attagccact gttttgaatc cacatactcc aatactcctg
     5101 aaatagttca ttatggacag cgcagaaaga gctggggaga attgtgaaat tgttatccgc
     5161 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5221 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5281 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5341 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5401 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5461 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5521 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5941 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6481 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6541 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6601 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6661 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6721 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6781 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6841 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6901 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6961 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7021 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7081 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7141 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7201 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7261 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     7321 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     7381 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     7441 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     7501 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     7561 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
     7621 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     7681 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcgc
     7741 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     7801 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     7861 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgcc
//
