LOCUS       sequence-106497-        7532 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-Ago2 S387A mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7532)
  AUTHORS   Horman SR, Janas MM, Litterst C, Wang B, MacRae IJ, Sever MJ, 
            Morrissey DV, Graves P, Luo B, Umesalma S, Qi HH, Miraglia LJ, 
            Novina CD, Orth AP
  TITLE     Akt-mediated phosphorylation of argonaute 2 downregulates cleavage 
            and upregulates translational repression of MicroRNA targets.
  JOURNAL   Mol Cell. 2013 May 9;50(3):356-67. doi: 
            10.1016/j.molcel.2013.03.015. Epub 2013 Apr 18.
  PUBMED    23603119
REFERENCE   2  (bases 1 to 7532)
  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.2013.03.015"; journalName: "Mol Cell"; date: 
            "2013-05-9- 9"; volume: "50"; issue: "3"; pages: "356-67"
COMMENT     Sequence Label: pGEX 4T-1 GST-Ago2 S387A
FEATURES             Location/Qualifiers
     source          1..7532
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2868..2877
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3582..3604)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3749..3767)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3835..3939
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3940..4008,4009..4800)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4158..4177)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4971..5559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5460..5479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5713..5730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5803..5880
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5881..6963
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5900..5919)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6976..6997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7012..7017,7018..7035,7036..7042)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7050..7066
                     /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)."
     primer_bind     7074..7090
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7074..7090
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(7086..7532,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(7106..7123)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7106..7122)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7115..7137)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccccgga attctactcg ggagccggcc
      961 ccgcacttgc acctcctgcg ccgccgcccc ccatccaagg atatgccttc aagcctccac
     1021 ctagacccga ctttgggacc tccgggagaa caatcaaatt acaggccaat ttcttcgaaa
     1081 tggacatccc caaaattgac atctatcatt atgaattgga tatcaagcca gagaagtgcc
     1141 cgaggagagt taacagggaa atcgtggaac acatggtcca gcactttaaa acacagatct
     1201 ttggggatcg gaagcccgtg tttgacggca ggaagaatct atacacagcc atgccccttc
     1261 cgattgggag ggacaaggtg gagctggagg tcacgctgcc aggagaaggc aaggatcgca
     1321 tcttcaaggt gtccatcaag tgggtgtcct gcgtgagctt gcaggcgtta cacgatgcac
     1381 tttcagggcg gctgcccagc gtcccttttg agacgatcca ggccctggac gtggtcatga
     1441 ggcacttgcc atccatgagg tacacccccg tgggccgctc cttcttcacc gcgtccgaag
     1501 gctgctctaa ccctcttggc gggggccgag aagtgtggtt tggcttccat cagtccgtcc
     1561 ggccttctct ctggaaaatg atgctgaata ttgatgtgtc agcaacagcg ttttacaagg
     1621 cacagccagt aatcgagttt gtttgtgaag ttttggattt taaaagtatt gaagaacaac
     1681 aaaaacctct gacagattcc caaagggtaa agtttaccaa agaaattaaa ggtctaaagg
     1741 tggagataac gcactgtggg cagatgaaga ggaagtaccg cgtctgcaat gtgacccggc
     1801 ggcccgccag tcaccaaaca ttcccgctgc agcaggagag cgggcagacg gtggagtgca
     1861 cggtggccca gtatttcaag gacaggcaca agttggttct gcgctacccc cacctcccat
     1921 gtttacaagt cggacaggag cagaaacaca cctaccttcc cctggaggtc tgtaacattg
     1981 tggcaggaca aagatgtatt aaaaaattaa cggacaatca gacctcaacc atgatcagag
     2041 cgactgctag gtcggcgccc gatcggcaag aagagattag caaattgatg cgaagtgcag
     2101 ctttcaacac agatccatac gtccgtgaat ttggaatcat ggtcaaagat gagatgacag
     2161 acgtgactgg gcgggtgctg cagccgccct ccatcctcta cgggggcagg aataaagcta
     2221 ttgcgacccc tgtccagggc gtctgggaca tgcggaacaa gcagttccac acgggcatcg
     2281 agatcaaggt gtgggccatt gcgtgcttcg ccccccagcg ccagtgcacg gaagtccatc
     2341 tgaagtcctt cacagagcag ctcagaaaga tctcgagaga cgccggcatg cccatccagg
     2401 gccagccgtg cttctgcaaa tacgcgcagg gggcggacag cgtggagccc atgttccggc
     2461 acctgaagaa cacgtatgcg ggcctgcagc tggtggtggt catcctgccc ggcaagacgc
     2521 ccgtgtacgc cgaggtcaag cgtgtgggag acacggtgct ggggatggcc acgcagtgcg
     2581 tgcagatgaa gaacgtgcag aggaccacgc cacagaccct gtccaacctc tgcctgaaga
     2641 tcaacgtcaa gctgggaggc gtgaacaaca tcctgctgcc ccagggcagg ccgccggtgt
     2701 tccagcagcc cgtcatcttt ctgggagcag acgtcactca cccccccgcc ggggatggga
     2761 agaagccctc cattgccgcc gtggtgggca gcatggacgc ccaccccaat cgctactgcg
     2821 ccaccgtgcg tgtgcagcag caccggcagg agatcataca agacctggcc gccatggtcc
     2881 gcgagctcct catccagttc tacaagtcca cgcgcttcaa gcccacccgc atcatcttct
     2941 accgcgacgg tgtctctgaa ggccagttcc agcaggttct ccaccacgag ttgctggcca
     3001 tccgtgaggc ctgtatcaag ctagaaaaag actaccagcc agggatcacc ttcatcgtgg
     3061 tgcagaagag gcaccacacc cggctcttct gcactgacaa gaacgagcgg gttgggaaaa
     3121 gtggaaacat tccagcaggc acgactgtgg acacgaaaat cacccacccc acagagttcg
     3181 acttctacct gtgtagtcac gctggcatcc aggggacaag caggccttcg cactatcacg
     3241 tcctctggga cgacaatcgt ttctcctctg atgagctgca gatcctaacc taccagctgt
     3301 gtcacaccta cgtgcgctgc acacgctccg tgtccatccc agcgccagca tactacgctc
     3361 acctggtggc cttccgggcc aggtaccacc tggtggataa ggaacatgac agtgctgaag
     3421 gaagccatac ctctgggcag agtaacgggc gagaccacca agcactggcc aaggcggtcc
     3481 aggttcacca agacactctg cgcaccatgt actttgcttg agcggccgca tcgtgactga
     3541 ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3601 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3661 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     3721 ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3781 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3841 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3901 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3961 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4021 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4081 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4141 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     4201 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4261 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4321 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4381 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4441 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     4501 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4561 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4621 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4681 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4741 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4801 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4861 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4921 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4981 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5041 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5101 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     5161 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5221 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5281 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5341 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5401 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5461 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5521 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5581 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5641 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5701 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5761 ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggtgc
     5821 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     5881 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     5941 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     6001 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     6061 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     6121 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     6181 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     6241 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     6301 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     6361 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     6421 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     6481 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     6541 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     6601 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     6661 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     6721 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     6781 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     6841 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     6901 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6961 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7021 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7081 cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     7141 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     7201 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     7261 ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc
     7321 ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc
     7381 ccatctacac caacgtaacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga
     7441 atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc
     7501 agacgcgaat tatttttgat ggcgttggaa tt
//
