LOCUS       sequence-356771-        5656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsNot9-Y203AR205AH208AR244A-shRNA1res.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5656)
  AUTHORS   Chen Y, Boland A, Kuzuoglu-Ozturk D, Bawankar P, Loh B, Chang CT, 
            Weichenrieder O, Izaurralde E
  TITLE     A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct 
            links between miRNA target recognition and silencing.
  JOURNAL   Mol Cell. 2014 Jun 5;54(5):737-50. doi: 
            10.1016/j.molcel.2014.03.034. Epub 2014 Apr 24.
  PUBMED    24768540
REFERENCE   2  (bases 1 to 5656)
  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.2014.03.034"; journalName: "Mol Cell"; date: 
            "2014-06-5- 5"; volume: "54"; issue: "5"; pages: "737-50"
COMMENT     Sequence Label: pT7-EGFP-C1-HsNot9-Y203AR205AH208AR244A-shRNA1...
FEATURES             Location/Qualifiers
     source          1..5656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     553..572
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        553..571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      599..608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(605..607,608..610,611..1321)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(605..607,608..610,611..1321)
                     /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(650..671)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(911..930)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1258..1279
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2384..2505
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2421..2440)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2475..2494
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2512..2967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2649..2670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2994..3098
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3100..3457
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3100..3291
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3123..3143)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3308..3443
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3492..4286
                     /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             3492..4286
                     /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(3546..3565)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4156..4175
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4474..4493)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4518..4565
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4894..5482
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5383..5402
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      541 gcttatcgaa attaatacga ctcactatag ggagacccaa gctctagcgc taccggtcgc
      601 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      661 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      721 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      781 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      841 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      901 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      961 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1021 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1081 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1141 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1201 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1261 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1321 gtccggactc agatctcgag ctcaagcttc gaattctcac agcctggcga cggctgcgcc
     1381 tgtgcctact acactggcac aagtggatag agaaaagatc tatcagtgga tcaatgagct
     1441 gtccagtcct gagactaggg aaaatgcttt gctggagcta agtaagaagc gagaatctgt
     1501 tcctgacctt gcacccatgt tatggcacag tttcggtact attgcagcac ttttacagga
     1561 aattgtaaat atttatccat ctatcaaccc acccaccttg acagcacacc agtctaacag
     1621 agtttgcaat gctctggcat tactgcaatg tgtagcatca catccagaaa ccaggtcagc
     1681 gtttctcgca gcacacatcc cacttttttt gtaccccttt ttgcacactg tcagcaaaac
     1741 acgtcccttt gagtatctcc ggctcaccag ccttggagtt attggggccc tggtgaaaac
     1801 agatgaacaa gaagtaatca actttttatt aacaacagaa attatccctt tatgtttgcg
     1861 aattatggaa tctggaagtg aactttctaa aacagttgcc acattcatcc tccagaagat
     1921 cttgttagat gacactggtt tggcttatat atgtcagacg gcagaggcat tctccgcagt
     1981 tgccatgatc ttgggtaaga tggtcctgca gctatccaaa gagccttctg cccgtctgct
     2041 gaagcatgta gtgagatgtt accttcgact ttcagataac cccgctgcac gtgaagcact
     2101 cagacagtgc ctccctgacc agctgaaaga cacaaccttc gcccaggtgc taaaagatga
     2161 caccaccacg aaacgctggc ttgcacaact ggtgaagaac ctgcaagagg gccaggtcac
     2221 cgatccccgg ggtatccccc tgccccctca gtgaggatcc accggatcta gataactgat
     2281 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     2341 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     2401 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2461 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa
     2521 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2581 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2641 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2701 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3001 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3061 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     3121 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3181 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3241 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3301 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3361 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3421 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     3481 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3541 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3601 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3661 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3721 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3781 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3841 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3901 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3961 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     4021 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4081 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4141 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4201 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4261 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4321 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4381 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4441 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     4501 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     4561 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     4621 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     4681 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     4741 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4801 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4861 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4921 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4981 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5041 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5101 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5161 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5221 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5281 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5341 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5401 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5461 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5521 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5581 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     5641 catatatgga gttccg
//
