LOCUS       sequence-209057-        7936 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 7936)
  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 7936)
  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..7936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1592..1606
                     /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        1790..2289
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1825..1848)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2222..2241
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2310..2909
                     /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(2310..2329)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2806..2826
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2970..3537
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2987..3539
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3131..3148)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3358..3377
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3551..4246
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     LTR             4298..4812
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(4981..4997)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4981..4997)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4994..5016)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5005..5021
                     /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(5029..5035,5036..5053,5054..5059))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5074..5095
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5212..5229)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5383..5971)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5463..5482)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6142..6933,6934..7002))
                     /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     6765..6784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7003..7107)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7175..7193
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7231..7253)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7353..7372
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7566..7588
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7716..7735
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7935..7936,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ctagggataa cagggtaatt
     1441 gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc
     1501 tgggattact tcgacttctt aacccaacag aaggctcgag aaggtatatt gctgttgaca
     1561 gtgagcgcag gaattataat gcttatctat agtgaagcca cagatgtata gataagcatt
     1621 ataattccta tgcctactgc ctcggacttc aaggggctag aattcgagca attatcttgt
     1681 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     1741 ggtataaatt aaatcacttt tttcaattga cgcgtgatct aattctaccg ggtaggggag
     1801 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     1861 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     1921 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     1981 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
     2041 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     2101 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     2161 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     2221 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     2281 ggcctttcga cctgcagccc aagcttacca tgaccgagta caagcccacg gtgcgcctcg
     2341 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     2401 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     2461 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     2521 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     2581 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     2641 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     2701 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     2761 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     2821 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     2881 ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg
     2941 aaaggagcgc acgaccccat gcatccaatt ccgccccccc cccctaacgt tactggccga
     3001 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     3061 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     3121 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     3181 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     3241 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     3301 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     3361 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     3421 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     3481 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat
     3541 ggccacaacc atggcccagt ccaagcacgg cctgaccaag gagatgacca tgaagtaccg
     3601 catggagggc tgcgtggacg gccacaagtt cgtgatcacc ggcgagggca tcggctaccc
     3661 cttcaagggc aagcaggcca tcaacctgtg cgtggtggag ggcggcccct tgcccttcgc
     3721 cgaggacatc ttgtccgccg ccttcatgta cggcaaccgc gtgttcaccg agtaccccca
     3781 ggacatcgtc gactacttca agaactcctg ccccgccggc tacacctggg accgctcctt
     3841 cctgttcgag gacggcgccg tgtgcatctg caacgccgac atcaccgtga gcgtggagga
     3901 gaactgcatg taccacgagt ccaagttcta cggcgtgaac ttccccgccg acggccccgt
     3961 gatgaagaag atgaccgaca actgggagcc ctcctgcgag aagatcatcc ccgtgcccaa
     4021 gcagggcatc ttgaagggcg acgtgagcat gtacctgctg ctgaaggacg gtggccgctt
     4081 gcgctgccag ttcgacaccg tgtacaaggc caagtccgtg ccccgcaaga tgcccgactg
     4141 gcacttcatc cagcacaagc tgacccgcga ggaccgcagc gacgccaaga accagaagtg
     4201 gcacctgacc gagcacgcca tcgcctccgg ctccgccttg ccctgaagat catcgataaa
     4261 ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt
     4321 tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaatacata actgagaata
     4381 gagaagttca gatcaaggtt aggaacagag agacagcaga atatgggcca aacaggatat
     4441 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt
     4501 cccgccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga
     4561 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg
     4621 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc
     4681 cgatagactg cgtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg
     4741 acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg
     4801 ggggtctttc atgggtaaca gtttcttgaa gttggagaac aacattctga gggtaggagt
     4861 cgaatattaa gtaatcctga ctcaattagc cactgttttg aatccacata ctccaatact
     4921 cctgaaatag ttcattatgg acagcgcaga agagctgggg agaattaatt cgtaatcatg
     4981 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5041 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5101 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5221 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5281 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5341 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5401 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5461 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5521 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5581 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5641 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5701 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5761 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5821 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5881 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5941 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6001 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6061 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6121 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6181 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6241 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6301 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6361 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6421 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6481 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6541 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6601 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6661 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6721 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6781 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6841 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6901 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6961 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7021 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7081 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7141 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7201 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     7261 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7321 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     7381 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     7441 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     7501 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     7561 gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga atggtgcatg caaggagatg
     7621 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     7681 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     7741 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggcgatta
     7801 gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac tcaacaatat
     7861 caccagctga agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc
     7921 cagaaaaagg ggggaa
//
