LOCUS       sequence-290902-        6174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsRck-RFQNRtoEDAAA-shRNAres.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6174)
  AUTHORS   Kuzuoglu-Ozturk D, Bhandari D, Huntzinger E, Fauser M, Helms S, 
            Izaurralde E
  TITLE     miRISC and the CCR4-NOT complex silence mRNA targets independently 
            of 43S ribosomal scanning.
  JOURNAL   EMBO J. 2016 Jun 1;35(11):1186-203. doi: 10.15252/embj.201592901. 
            Epub 2016 Mar 23.
  PUBMED    27009120
REFERENCE   2  (bases 1 to 6174)
  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: 
            "10.15252/embj.201592901"; journalName: "EMBO J"; date: "2016-06-1- 
            1"; volume: "35"; issue: "11"; pages: "1186-203"
COMMENT     Sequence Label: pT7-EGFP-C1-HsRCK-RFQNRtoEDAAA-shRNAres_AC
FEATURES             Location/Qualifiers
     source          1..6174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     613..632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        613..631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      659..668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(665..667,668..670,671..1381)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(710..731)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(971..990)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1318..1339
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2962..3083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2999..3018)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3053..3072
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3090..3545)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3227..3248)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3439..3458
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3572..3676
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3678..4035
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3678..3869
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3701..3721)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3886..4021
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4070..4864
                     /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             4070..4864
                     /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(4124..4143)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4734..4753
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5052..5071)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5096..5143
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5472..6060
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5961..5980
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      601 gcttatcgaa attaatacga ctcactatag ggagacccaa gctctagcgc taccggtcgc
      661 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      721 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      781 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      841 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      901 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      961 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1021 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1081 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1141 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1201 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1261 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1321 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1381 gtccggactc agatctcgag ctatgggtct gtccagtcaa aatggtcagc tgagaggccc
     1441 tgtgaaaccc actggtggcc ctggaggagg gggcacacag acacagcaac agatgaacca
     1501 gctgaaaaac accaacacaa tcaataatgg cactcagcag caagcacaga gtatgaccac
     1561 cactattaaa cctggtgatg actggaaaaa gactttaaaa ctccctccaa aggatctaag
     1621 aatcaaaact tcggatgtga cctccacaaa aggaaatgag tttgaagatt actgtttgaa
     1681 agaggagtta ctgatgggaa ttgatgaaat gggctgggaa aagccatctc ctattcagga
     1741 ggagagcatt cccattgctt tatctggtag ggatatctta gctagagcaa aaaatggaac
     1801 aggcaagagc ggtgcctacc tcattccctt acttgaacgg ctagacctga agaaggacaa
     1861 tatacaagca atggtgattg ttcccactag agaacttgct ctacaggtca gtcaaatttg
     1921 catccaggtc agcaaacaca tgggaggggc caaagtgatg gcaaccacag gaggaaccaa
     1981 tttacgagat gacataatga ggcttgatga tacagtgcac gtggtgattg ctacccctgg
     2041 gagaatcctg gatcttatta agaaaggagt agcaaaggtt gatcatgtcc agatgatagt
     2101 attggatgag gcagataagt tgctgtcaca ggattttgtg cagataatgg aggatattat
     2161 tctcacgcta cctaaaaaca ggcagatttt actatattcc gctactttcc ctcttagtgt
     2221 acagaagttc atgaattccc atttacaaaa gccatacgaa attaacctga tggaggaact
     2281 aactctgaag ggagtaaccc agtactacgc atatgtaact gagcgccaaa aagtacactg
     2341 cctcaacaca cttttctcca ggcttgcaat agcccagtcg atcattttct gtaactcctc
     2401 tcagcgagtt gaattgctag ccaagaagat ttctcaactg ggttattctt gcttctatat
     2461 tcatgctaaa atgaggcagg aacatcgaaa tcgtgtattt catgatttcc gaaatggctt
     2521 atgcgccaat cttgtttgca ctgatctgtt tacccgaggt attgatatac aagctgtgaa
     2581 tgtggtaata aactttgatt tcccaaagct ggcagagacc tatctccatc gtattggaag
     2641 atcaggtcgc tttggtcatc ttggcttagc catcaacttg atcacatatg atgatcgctt
     2701 caacctgaaa agtattgagg agcagctggg aacagaaatt aaacctattc cgagcaacat
     2761 tgataagagc ctgtatgtgg cagaatacca cagcgagcct gtagaagatg agaaacctta
     2821 accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca taccacattt
     2881 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2941 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     3001 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3061 tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt
     3121 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3181 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3241 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3301 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3361 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3421 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3481 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3541 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3601 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3661 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3721 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3781 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3841 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3901 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3961 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4021 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4081 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4141 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4201 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4261 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4321 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4381 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4441 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4501 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4561 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4621 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4681 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4741 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4801 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4861 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4921 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4981 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     5041 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     5101 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     5161 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     5221 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     5281 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     5341 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5401 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5461 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5521 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5581 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     5641 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5701 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5761 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5821 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5881 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5941 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6001 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6061 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6121 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat
//
