LOCUS       sequence-328518-        8682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Drosha-FLAG-mEGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8682)
  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 8682)
  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: pCK-Drosha(UniProt)-FLAG-mEGFP
FEATURES             Location/Qualifiers
     source          1..8682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        57..436
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        437..640
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     591..611
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     637..661
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          776..1601
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             5837..5860
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5861..5870
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5867..5869,5870..5872,5873..6586)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5912..5933)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     6520..6541
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6656..6673)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6662..6869
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(6878..7672)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(6878..7672)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6989..7008)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7599..7618
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      8084..8672
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8573..8592
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg cgcgttgaca
       61 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      121 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      181 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      241 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      301 gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      361 ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt
      421 catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt
      481 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      541 ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg
      601 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
      661 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      721 cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag
      781 taccgcctat agactctata ggcacacccc tttggctctt atgcatgcta tactgttttt
      841 ggcttggggc ctatacaccc ccgcttcctt atgctatagg tgatggtata gcttagccta
      901 taggtgtggg ttattgacca ttattgacca ctcccctatt ggtgacgata ctttccatta
      961 ctaatccata acatggctct ttgccacaac tatctctatt ggctatatgc caatactctg
     1021 tccttcagag actgacacgg actctgtatt tttacaggat ggggtcccat ttattattta
     1081 caaattcaca tatacaacaa cgccgtcccc cgtgcccgca gtttttatta aacatagcgt
     1141 gggatctcca cgcgaatctc gggtacgtgt tccggacatg ggctcttctc cggtagcggc
     1201 ggagcttcca catccgagcc ctggtcccat gcctccagcg gctcatggtc gctcggcagc
     1261 tccttgctcc taacagtgga ggccagactt aggcacagca caatgcccac caccaccagt
     1321 gtgccgcaca aggccgtggc ggtagggtat gtgtctgaaa atgagctcgg agattgggct
     1381 cgcaccgctg acgcagatgg aagacttaag gcagcggcag aagaagatgc aggcagctga
     1441 gttgttgtat tctgataaga gtcagaggta actcccgttg cggtgctgtt aacggtggag
     1501 ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg ccaccagaca taatagctga
     1561 cagactaaca gactgttcct ttccatgggt cttttctgca gtcaccgtcc ttgacacgaa
     1621 gcttggtacc gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca
     1681 cagtggcggc cgcgggaatt cgattcgagg taccatgatg cagggaaaca catgtcacag
     1741 aatgtcgttc cacccgggac gagggtgtcc ccgaggacga ggaggacatg gagccagacc
     1801 ctcagcacca tcctttaggc cccaaaatct gaggctgctt caccctcagc agcctcctgt
     1861 gcaatatcaa tatgaacctc caagtgcccc ttccaccact ttctcaaact ctccagcccc
     1921 caattttctc cctccacgac cagactttgt acccttcccc ccacccatgc ctccgtcagc
     1981 gcaaggccct cttcccccct gcccaatcag gccgcctttc cccaaccacc agatgaggca
     2041 ccccttccca gttcctcctt gttttcctcc catgccacca ccaatgcctt gtcctaataa
     2101 ccccccagtc cctggggcac ctcctggaca aggcactttc cccttcatga tgccccctcc
     2161 ctccatgcct catcccccgc cccctccagt catgccgcag caggttaatt atcagtaccc
     2221 tccgggctat tctcaccaca acttcccacc tcccagtttt aatagtttcc agaacaaccc
     2281 tagttctttc ctgcccagtg ctaataacag cagtagtcct catttcagac atctccctcc
     2341 atacccactc ccaaaggctc ccagtgagag aaggtcccca gaaaggctga aacactatga
     2401 tgaccacagg caccgagatc acagtcatgg gcgaggtgag aggcatcggt ccctggatcg
     2461 gcgggagcga ggccgcagtc ccgacaggag aagacaagac agccggtaca gatctgatta
     2521 tgaccgaggg agaacaccat ctcgccaccg cagctacgaa cggagcagag agcgagaacg
     2581 ggagagacac aggcatcgag acaaccgaag atcaccatct ctggaaaggt cctacaaaaa
     2641 agagtataag agatctggaa ggagttacgg tttatcggtt gttcctgaac ctgctggatg
     2701 cacaccagaa ttacctgggg agattattaa aaatacagat tcttgggccc cacccctgga
     2761 gattgtgaat catcgctccc caagtaggga gaagaagaga gctcgttggg aggaagaaaa
     2821 agaccgttgg agtgacaacc agagttctgg caaagacaag aactatacct caatcaagga
     2881 aaaagagccc gaggagacca tgcctgacaa gaatgaggag gaagaagaag aacttcttaa
     2941 gcctgtgtgg attcgatgca ctcattcaga aaactactac tccagtgacc ccatggatca
     3001 ggtgggagat tctacagtgg ttggaacgag taggcttcgt gacttatatg acaaatttga
     3061 ggaggagttg gggagcaggc aagaaaaggc caaagctgct cggcctccgt gggaacctcc
     3121 aaagacgaag ctcgatgaag atttagagag ttccagtgaa tccgagtgtg agtctgatga
     3181 ggacagcacc tgttctagca gctcagactc tgaagttttt gacgttattg cagaaatcaa
     3241 acgcaaaaag gcccaccctg accgacttca tgatgaactt tggtacaacg atccaggcca
     3301 gatgaatgat ggaccactct gcaaatgcag cgcaaaggca agacgcacag gaattaggca
     3361 cagcatttat cctggagaag aggccatcaa gccctgtcgt cctatgacca acaatgctgg
     3421 cagacttttc cactaccgga tcacagtctc cccgcctacg aactttttaa ctgacaggcc
     3481 aactgttata gaatacgatg atcacgagta tatctttgaa ggattttcta tgtttgcaca
     3541 tgcccccctg accaatattc cactgtgtaa agtaattaga ttcaacatag actacacgat
     3601 tcatttcatt gaagagatga tgccggagaa tttttgtgtg aaagggcttg aactcttttc
     3661 actgttccta ttcagagata ttttggaatt atatgactgg aatcttaaag gtcctttgtt
     3721 tgaagacagc cctccctgct gcccaagatt tcatttcatg ccacgttttg taagatttct
     3781 tccagatgga ggaaaggaag tgctgtccat gcaccagatt ctcctgtact tgttaaggtg
     3841 cagcaaagcc ctggtgcctg aggaggagat tgccaatatg cttcagtggg aggagctgga
     3901 gtggcagaaa tatgcagaag aatgcaaagg catgattgtt accaaccctg ggacgaaacc
     3961 aagctctgtc cgtatcgatc aactggatcg tgaacagttc aaccccgatg tgattacttt
     4021 tccgattatc gtccactttg ggatacgccc tgcacagttg agttatgcag gagacccaca
     4081 gtaccaaaaa ctgtggaaga gttatgtgaa acttcgccac ctcctagcaa atagtcccaa
     4141 agtcaaacaa actgacaaac agaagctggc acagagggag gaagccctcc aaaaaatacg
     4201 gcagaagaat acaatgagac gagaagtaac ggtggagcta agtagccaag gattctggaa
     4261 aactggcatc cgttctgatg tctgtcagca tgcaatgatg ctacctgttc tgacccatca
     4321 tatccgctac caccaatgcc taatgcattt ggacaagttg ataggatata ctttccaaga
     4381 tcgttgtctg ttgcagctgg ccatgactca tccaagtcat catttaaatt ttggaatgaa
     4441 tcctgatcat gccaggaatt cattatctaa ctgtggaatt cggcagccca aatacggaga
     4501 cagaaaagtt catcacatgc acatgcggaa gaaagggatt aacaccttga taaatatcat
     4561 gtcacgcctt ggccaagatg acccaactcc ctcgaggatt aaccacaatg aacggttgga
     4621 attcctgggt gatgctgttg ttgaatttct gaccagcgtc catttgtact atttgtttcc
     4681 tagtctggaa gaaggaggat tagcaaccta tcggactgcc attgttcaga atcagcacct
     4741 tgccatgcta gcaaagaaac ttgaactgga tcgatttatg ctgtatgctc acgggcctga
     4801 cctttgtaga gaatcggacc ttcgacatgc aatggccaat tgttttgaag cgttaatagg
     4861 agctgtttac ttggagggaa gcctggagga agccaagcag ttatttggac gcttgctctt
     4921 taatgatccg gacctgcgcg aagtctggct caattatcct ctccacccac tccaactaca
     4981 agagccaaat actgatcgac aacttattga aacttctccg gttctacaaa aacttactga
     5041 gtttgaagaa gcaattggag taatttttac tcatgttcga cttctggcaa gggcattcac
     5101 attgagaact gtgggattta accatctgac cctaggccac aatcagagaa tggaattcct
     5161 aggtgactcc ataatgcaac tggtagccac agagtactta ttcattcatt tcccagatca
     5221 tcatgaagga cacttaactt tgttgcgaag ctctttggtg aataatagaa ctcaggccaa
     5281 ggtagcggag gagctgggca tgcaggagta cgccataacc aacgacaaga ccaagaggcc
     5341 tgtggcgctt cgcaccaaga ccttggcgga ccttttggaa tcatttattg cagcgctgta
     5401 cattgataag gatttggaat atgttcatac tttcatgaat gtctgcttct ttccacgatt
     5461 gaaagagttc attttgaatc aggattggaa tgaccccaaa tcccagcttc agcagtgttg
     5521 cttgacactt aggacagaag gaaaagagcc agacattcct ctgtacaaga ctctgcagac
     5581 agtgggccca tcccatgccc gaacctacac tgtggctgtt tatttcaagg gagaaagaat
     5641 aggctgtggg aaaggaccaa gtattcagca agcggaaatg ggagcagcaa tggatgcgct
     5701 tgaaaaatat aattttcccc agatggccca tcagaagcgg ttcatcgaac ggaagtacag
     5761 acaagagtta aaagaaatga ggtgggaaag agagcatcaa gagagagagc cagatgagac
     5821 tgaagacatc aagaaagact acaaagacga tgacgataaa gccaccatgg tgagcaaggg
     5881 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     5941 ccacaagttc agcgtgcgcg gcgagggcga gggcgatgcc accaacggca agctgaccct
     6001 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     6061 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     6121 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atctccttca aggacgacgg
     6181 cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     6241 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     6301 cttcaacagc cacaacgtct atatcacggc cgacaagcag aagaacggca tcaaggcgaa
     6361 cttcaagatc cgccacaacg tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     6421 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     6481 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     6541 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaataatcta gagacaatca
     6601 ctagtgaatt cgcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     6661 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     6721 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     6781 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     6841 tgggaagaca atagcaggca tgctggggag tcgaaattca gaagaactcg tcaagaaggc
     6901 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     6961 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     7021 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     7081 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     7141 tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     7201 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     7261 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     7321 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     7381 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     7441 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tgcagttcat
     7501 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     7561 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     7621 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     7681 atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca
     7741 agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg
     7801 gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc
     7861 cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt
     7921 agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgaaaagga
     7981 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     8041 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     8101 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     8161 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     8221 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     8281 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     8341 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcacag cggtcgggct
     8401 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     8461 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     8521 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     8581 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     8641 gatgctcgtc aggggggcgg agcctatgga aaaacgccag ca
//
