LOCUS       sequence-290899-        6188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsRck-K135A-shRNAres.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6188)
  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 6188)
  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-K135A-shRNAres_AC
FEATURES             Location/Qualifiers
     source          1..6188
                     /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    2976..3097
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3013..3032)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3067..3086
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3104..3559)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3241..3262)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3453..3472
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3586..3690
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3692..4049
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3692..3883
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3715..3735)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3900..4035
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4084..4878
                     /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             4084..4878
                     /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(4138..4157)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4748..4767
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5066..5085)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5110..5157
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5486..6074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5975..5994
                     /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 agatctatgg gtctgtccag tcaaaatggt cagctgagag gccctgtgaa
     1441 acccactggt ggccctggag gagggggcac acagacacag caacagatga accagctgaa
     1501 aaacaccaac acaatcaata atggcactca gcagcaagca cagagtatga ccaccactat
     1561 taaacctggt gatgactgga aaaagacttt aaaactccct ccaaaggatc taagaatcaa
     1621 aacttcggat gtgacctcca caaaaggaaa tgagtttgaa gattactgtt tgaaacggga
     1681 gttactgatg ggaatttttg aaatgggctg ggaaaagcca tctcctattc aggaggagag
     1741 cattcccatt gctttatctg gtagggatat cttagctaga gcaaaaaatg gaacaggcgc
     1801 aagcggtgcc tacctcattc ccttacttga acggctagac ctgaagaagg acaatataca
     1861 agcaatggtg attgttccca ctagagaact tgctctacag gtcagtcaaa tttgcatcca
     1921 ggtcagcaaa cacatgggag gggccaaagt gatggcaacc acaggaggaa ccaatttacg
     1981 agatgacata atgaggcttg atgatacagt gcacgtggtg attgctaccc ctgggagaat
     2041 cctggatctt attaagaaag gagtagcaaa ggttgatcat gtccagatga tagtattgga
     2101 tgaggcagat aagttgctgt cacaggattt tgtgcagata atggaggata ttattctcac
     2161 gctacctaaa aacaggcaga ttttactata ttccgctact ttccctctta gtgtacagaa
     2221 gttcatgaat tcccatttac aaaagccata cgaaattaac ctgatggagg aactaactct
     2281 gaagggagta acccagtact acgcatatgt aactgagcgc caaaaagtac actgcctcaa
     2341 cacacttttc tccaggcttc agataaacca gtcgatcatt ttctgtaact cctctcagcg
     2401 agttgaattg ctagccaaga agatttctca actgggttat tcttgcttct atattcatgc
     2461 taaaatgagg caggaacatc gaaatcgtgt atttcatgat ttccgaaatg gcttatgccg
     2521 caatcttgtt tgcactgatc tgtttacccg aggtattgat atacaagctg tgaatgtggt
     2581 aataaacttt gatttcccaa agctggcaga gacctatctc catcgtattg gaagatcagg
     2641 tcgctttggt catcttggct tagccatcaa cttgatcaca tatgatgatc gcttcaacct
     2701 gaaaagtatt gaggagcagc tgggaacaga aattaaacct attccgagca acattgataa
     2761 gagcctgtat gtggcagaat accacagcga gcctgtagaa gatgagaaac cttaacaatt
     2821 ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc tagataactg atcataatca
     2881 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     2941 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     3001 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3061 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat
     3121 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3181 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3241 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3301 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3361 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3421 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3481 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3541 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3601 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3661 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     3721 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3781 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3841 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3901 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3961 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4021 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     4081 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4141 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4201 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4261 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4321 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4381 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4441 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4501 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4561 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     4621 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4681 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4741 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4801 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4861 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4921 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4981 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     5041 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     5101 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     5161 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     5221 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     5281 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     5341 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5401 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5461 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5521 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5581 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5641 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5701 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5761 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5821 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5881 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5941 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6001 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6061 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6121 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6181 ccatgcat
//
