LOCUS       sequence-206276-        7524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express DGCR8 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7524)
  AUTHORS   Chang TC, Pertea M, Lee S, Salzberg SL, Mendell JT
  TITLE     Genome-wide annotation of microRNA primary transcript structures 
            reveals novel regulatory mechanisms.
  JOURNAL   Genome Res. 2015 Sep;25(9):1401-9. doi: 10.1101/gr.193607.115.
  PUBMED    26290535
REFERENCE   2  (bases 1 to 7524)
  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: "10.1101/gr.193607";
            journalName: "Genome Res"; date: "2015-09"; volume: "25"; issue: 
            "9"; pages: "1401-9"
COMMENT     Sequence Label: pcDNA5/FLAG-HA-DGCR8
FEATURES             Location/Qualifiers
     source          1..7524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             926..949
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             950..976
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    992..1016
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3476..3493)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3482..3706
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3839..3858)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3990..4037
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4045..5067
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4045..5067
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5197..5318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5234..5253)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5288..5307
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5367..5383)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5367..5383)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5380..5402)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5391..5407
                     /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(5415..5421,5422..5439,5440..5445))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5460..5481
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5598..5615)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5769..6357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5849..5868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6528..7319,7320..7388))
                     /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     7151..7170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7389..7493)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggacta caaggacgac gatgacaagt acccttatga
      961 cgtgcccgat tacgctttga accagatatc aacaagtttg tacaaaaaag caggctccac
     1021 catgggaacc aattcagtcg acatggagac agatgagagc ccctctccgc tcccgtgtgg
     1081 gcccgcagga gaagcggtga tggagagccg agctcgcccc ttccaagcgc tgccccgtga
     1141 gcagtctcca ccacctcccc tgcaaacgtc cagtggtgca gaggtaatgg acgttggctc
     1201 tggtggtgat ggacagtccg aactccctgc tgaggacccc ttcaacttct acggagcttc
     1261 tcttctctcc aaaggatcct tctctaaggg ccgcctcctc atagacccga actgtagtgg
     1321 ccacagcccg cgcaccgccc ggcacgcacc tgcggtccgg aagttctccc ctgaccttaa
     1381 gttgcttaag gatgtaaaga ttagcgtgag ctttaccgag agctgcagga gtaaggacag
     1441 gaaggtgctg tacacaggag cagagcgcga cgtgcgggcg gagtgcggtc tgctccttag
     1501 ccctgtcagt ggggacgtgc atgcttgtcc ctttggcggg agtgttggtg acggggtagg
     1561 catagggggt gagagtgctg ataagaagga tgaggagaat gagctggatc aggaaaagag
     1621 agtggagtat gcagtgctcg atgagttaga agattttact gacaatttgg agctagatga
     1681 agaaggagca ggcgggttca cggctaaagc aatcgttcag agagacagag tggatgaaga
     1741 ggccttgaat ttcccctacg aggatgactt tgacaacgat gtggatgctc tgctggaaga
     1801 aggcctttgt gcccccaaaa agaggcgaac agaggaaaaa tatggcggag acagcgacca
     1861 tccgtccgat ggagagacaa gtgtgcagcc gatgatgacc aagattaaaa cagtgctcaa
     1921 aagtcgtggc cgcccaccta cagagccgct gcccgacggg tggatcatga cattccataa
     1981 ctctggagtc ccggtgtacc tacacagaga gtctcgggtg gtcacctggt ccaggccata
     2041 cttcttggga acgggaagca tacggaaaca cgaccctcct ctgagtagca tcccttgtct
     2101 gcattataag aaaatgaagg acaacgagga acgggagcaa agcagtgacc tcacccctag
     2161 tggggatgtg tcccccgtca agcccctgag ccgatctgca gagctggagt ttcccctgga
     2221 tgagcctgac tctatgggtg ctgacccggg gcccccggac gagaaagacc cactaggggc
     2281 tgaggcagcc cctggggccc tggggcaggt gaaggccaaa gtcgaggtgt gcaaagatga
     2341 atccgttgat ctcgaggaat ttcgaagcta cctggagaag cgttttgact ttgagcaagt
     2401 tactgtgaaa aaattcagga cttgggctga gcggcggcaa ttcaatcggg aaatgaagcg
     2461 gaagcaggcg gagtccgaga ggcccatctt gccagccaat cagaagctca ttactttatc
     2521 agtgcaagat gcacccacaa agaaagagtt tgttattaac cccaacggga aatccgaggt
     2581 ctgcatcctg cacgagtaca tgcagcgtgt cctcaaggtc cgccctgtct ataatttctt
     2641 tgaatgtgag aacccaagtg agccttttgg tgcctcggtg accattgatg gtgtgactta
     2701 cggatctgga actgcaagca gcaaaaaact tgcgaagaat aaagctgccc gagctacact
     2761 ggaaatcctc atccctgact ttgttaaaca gacctctgaa gagaagccca aagacagtga
     2821 agaactcgag tattttaacc acatcagcat cgaggactcg cgggtctacg agctgaccag
     2881 caaggctggg ctgttgtctc catatcagat cctccacgag tgccttaaaa gaaaccatgg
     2941 gatgggtgac acgtctatca agtttgaagt ggttcctggg aaaaaccaga agagtgaata
     3001 cgtcatggcg tgtggcaagc acacagtgcg cgggtggtgt aagaacaaga gagttggaaa
     3061 gcagttagcc tcacagaaga tccttcagct gctgcaccca catgtcaaga actgggggtc
     3121 tttactgcgc atgtatggcc gtgagagcag caagatggtc aagcaggaga catcggacaa
     3181 gagtgtgatt gagctgcagc agtatgccaa gaagaacaag cccaacctgc acatcctcag
     3241 caagctccaa gaggagatga agaggctagc tgaggaaagg gaggagactc gaaagaagcc
     3301 caagatgtcc attgtggcgt ccgcccagcc tggcggtgag cccctgtgca ccgtggacgt
     3361 gtgaaagctt ggtaccgagc tcggatccac tagtccagtg tggtggaatt ctgcagatat
     3421 ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     3481 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3541 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3601 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3661 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     3721 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     3781 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3841 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3901 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3961 cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta
     4021 gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag
     4081 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     4141 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     4201 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     4261 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     4321 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     4381 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     4441 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     4501 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     4561 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     4621 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     4681 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     4741 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     4801 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     4861 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     4921 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     4981 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     5041 agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc
     5101 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5161 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     5221 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5281 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     5341 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5401 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5461 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5521 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5581 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5641 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5701 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5761 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5821 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5881 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5941 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6001 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6061 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6121 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6181 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6241 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6301 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6361 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6421 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6481 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6541 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6601 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6661 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6721 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6781 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6841 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6901 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6961 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7021 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7081 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7141 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7201 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7261 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7321 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7381 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7441 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7501 ccccgaaaag tgccacctga cgtc
//
