LOCUS       sequence-327565-        7615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express DGCR8 without tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  AUTHORS   Fang W, Bartel DP
  TITLE     MicroRNA Clustering Assists Processing of Suboptimal MicroRNA 
            Hairpins through the Action of the ERH Protein.
  JOURNAL   Mol Cell. 2020 Apr 16;78(2):289-302.e6. doi: 
            10.1016/j.molcel.2020.01.026.
  PUBMED    32302541
REFERENCE   2  (bases 1 to 7615)
  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.molcel.2020.01"; journalName: "Mol Cell"; date: 
            "2020-04-16- 16"; volume: "78"; issue: "2"; pages: "289-302.e6"
COMMENT     Sequence Label: pNoTag-DGCR8
FEATURES             Location/Qualifiers
     source          1..7615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      712..721
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3063..3087)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3140..3181
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     intron          3262..3491
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3474..3493
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3563..4137,4138..4140,4141..4149)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3730..3747)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3957..3976
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4169..4768
                     /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(4169..4188)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4665..4685
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5002..5123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5039..5058)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5224..5240)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5224..5240)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5237..5259)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5248..5264
                     /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(5272..5278,5279..5296,5297..5302))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5317..5338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5455..5472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5626..6214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5706..5725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6385..7176,7177..7245))
                     /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     7008..7027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7246..7350)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7425..7444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcgat ccatcgatag tgtggtggaa ttctgatcgc cgccgccatg
      721 gagacagatg agagcccctc tccgctcccg tgtgggcccg caggagaagc ggtgatggag
      781 agccgagctc gccccttcca agcgctgccc cgtgagcagt ctccaccacc tcccctgcaa
      841 acgtccagtg gtgcagaggt aatggacgtt ggctctggtg gtgatggaca gtccgaactc
      901 cctgctgagg accccttcaa cttctacgga gcttctcttc tctccaaagg atccttctct
      961 aagggccgcc tcctcataga cccgaactgt agtggccaca gcccgcgcac cgcccggcac
     1021 gcacctgcgg tccggaagtt ctcccctgac cttaagttgc ttaaggatgt aaagattagc
     1081 gtgagcttta ccgagagctg caggagtaag gacaggaagg tgctgtacac aggagcagag
     1141 cgcgacgtgc gggcggagtg cggtctgctc cttagccctg tcagtgggga cgtgcatgct
     1201 tgtccctttg gcgggagtgt tggtgacggg gtaggcatag ggggtgagag tgctgataag
     1261 aaggatgagg agaatgagct ggatcaggaa aagagagtgg agtatgcagt gctcgatgag
     1321 ttagaagatt ttactgacaa tttggagcta gatgaagaag gagcaggcgg gttcacggct
     1381 aaagcaatcg ttcagagaga cagagtggat gaagaggcct tgaatttccc ctacgaggat
     1441 gactttgaca acgatgtgga tgctctgctg gaagaaggcc tttgtgcccc caaaaagagg
     1501 cgaacagagg aaaaatatgg cggagacagc gaccatccgt ccgatggaga gacaagtgtg
     1561 cagccgatga tgaccaagat taaaacagtg ctcaaaagtc gtggccgccc acctacagag
     1621 ccgctgcccg acgggtggat catgacattc cataactctg gagtcccggt gtacctacac
     1681 agagagtctc gggtggtcac ctggtccagg ccatacttct tgggaacggg aagcatacgg
     1741 aaacacgacc ctcctctgag tagcatccct tgtctgcatt ataagaaaat gaaggacaac
     1801 gaggaacggg agcaaagcag tgacctcacc cctagtgggg atgtgtcccc cgtcaagccc
     1861 ctgagccgat ctgcagagct ggagtttccc ctggatgagc ctgactctat gggtgctgac
     1921 ccggggcccc cggacgagaa agacccacta ggggctgagg cagcccctgg ggccctgggg
     1981 caggtgaagg ccaaagtcga ggtgtgcaaa gatgaatccg ttgatctcga ggaatttcga
     2041 agctacctgg agaagcgttt tgactttgag caagttactg tgaaaaaatt caggacttgg
     2101 gctgagcggc ggcaattcaa tcgggaaatg aagcggaagc aggcggagtc cgagaggccc
     2161 atcttgccag ccaatcagaa gctcattact ttatcagtgc aagatgcacc cacaaagaaa
     2221 gagtttgtta ttaaccccaa cgggaaatcc gaggtctgca tcctgcacga gtacatgcag
     2281 cgtgtcctca aggtccgccc tgtctataat ttctttgaat gtgagaaccc aagtgagcct
     2341 tttggtgcct cggtgaccat tgatggtgtg acttacggat ctggaactgc aagcagcaaa
     2401 aaacttgcga agaataaagc tgcccgagct acactggaaa tcctcatccc tgactttgtt
     2461 aaacagacct ctgaagagaa gcccaaagac agtgaagaac tcgagtattt taaccacatc
     2521 agcatcgagg actcgcgggt ctacgagctg accagcaagg ctgggctgtt gtctccatat
     2581 cagatcctcc acgagtgcct taaaagaaac catgggatgg gtgacacgtc tatcaagttt
     2641 gaagtggttc ctgggaaaaa ccagaagagt gaatacgtca tggcgtgtgg caagcacaca
     2701 gtgcgcgggt ggtgtaagaa caagagagtt ggaaagcagt tagcctcaca gaagatcctt
     2761 cagctgctgc acccacatgt caagaactgg gggtctttac tgcgcatgta tggccgtgag
     2821 agcagcaaga tggtcaagca ggagacatcg gacaagagtg tgattgagct gcagcagtat
     2881 gccaagaaga acaagcccaa cctgcacatc ctcagcaagc tccaagagga gatgaagagg
     2941 ctagctgagg aaagggagga gactcgaaag aagcccaaga tgtccattgt ggcgtccgcc
     3001 cagcctggcg gtgagcccct gtgcaccgtg gacgtgtgag cggccgcact cgagatatct
     3061 agacccagct ttcttgtaca aagtggttga tatccagcac agtggcggcc gctcaagtct
     3121 agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac
     3181 gcgtacctga tccgcggccg catagataac tgatccagtg tgctggaatt aattcgctgt
     3241 ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa agcgggcatg acttctgcgc
     3301 taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc gcggtgatgc
     3361 ctttgagggt ggccgcgtcc atctggtcag aaaagacaat ctttttgttg tcaagcttga
     3421 ggtgtggcag gcttgagatc tggccataca cttgagtgac aatgacatcc actttgcctt
     3481 tctctccaca ggtgtccact cccaggtcca actgcaggtc gagcatgcat ctagggcggc
     3541 caattccgcc cctctccccc cccccctctc cctccccccc ccctaacgtt actggccgaa
     3601 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     3661 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     3721 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
     3781 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     3841 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     3901 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     3961 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     4021 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac
     4081 gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt
     4141 gccacaaccc acaaggagac gaccttccat gaccgagtac aagcccacgg tgcgcctcgc
     4201 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     4261 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
     4321 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     4381 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     4441 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     4501 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     4561 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     4621 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     4681 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     4741 gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
     4801 aaggagcgca cgaccccatg gctccgaccg aagccacccg gggcggcccc gccgaccccg
     4861 cacccgcccc cgaggcccac cgactctaga taactgatca taatcagcca taccacattt
     4921 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     4981 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     5041 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5101 tccaaactca tcaatgtatc ttaacgctcg agtgcattct agttgtggtt tgtccaaact
     5161 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     5221 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5281 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5341 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5401 aatcggccaa cgctcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5461 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5521 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5581 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5641 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5701 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5761 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5821 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5881 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5941 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6001 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6061 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6121 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6181 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6241 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6301 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6361 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6421 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6481 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6541 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6601 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6661 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6721 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6781 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6841 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6901 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6961 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7021 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7081 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7141 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7201 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7261 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7321 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7381 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     7441 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     7501 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
     7561 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgtt
//
