LOCUS       sequence-289316-        5656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmGW182_862-1199-del1004-1115.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5656)
  AUTHORS   Huntzinger E, Braun JE, Heimstadt S, Zekri L, Izaurralde E
  TITLE     Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated 
            gene silencing.
  JOURNAL   EMBO J. 2010 Dec 15;29(24):4146-60. doi: 10.1038/emboj.2010.274
  PUBMED    21063388
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: 
            "10.1038/emboj.2010"; journalName: "EMBO J"; date: "2010-12-15- 15";
            volume: "29"; issue: "24"; pages: "4146-60"
COMMENT     Sequence Label: pGEX-6P-1-DmGW182_862-1199-del1004-1115_L
FEATURES             Location/Qualifiers
     source          1..5656
                     /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..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1706..1728)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1873..1891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1959..2063
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2064..2132,2133..2924)
                     /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(2282..2301)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3095..3683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3584..3603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3837..3854
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3927..4004
                     /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             4005..5087
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4024..4043)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5100..5121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5136..5141,5142..5159,5160..5166)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5174..5190
                     /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     5198..5214
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5198..5214
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5210..5656,1..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(5230..5247)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5230..5246)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5239..5261)
                     /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 gaagttctgt tccaggggcc cctgggatcc tatcaaggag
      961 cgtccaatca acaatcccga ttaaatcagt ggaagcttcc agtattagat aaggagatca
     1021 actctgacag tacggaattt tctcgtgccc caggtgcaac gaaacaaaat ttgacggcca
     1081 acacaagcaa cataaactct ttgggtcttc aaaacgatag tacatggtca actggacgca
     1141 gtattggtga cggttggcct gatccctcat ctgataacga gaataaagac tggtctgttg
     1201 ctcagccaac ttcagcagca actgcttaca ctgatctggt ccaagagttt gagccaggca
     1261 agccatggaa gggttcacag atcaaaagca tagaagatga tcccagcatt acaccaggaa
     1321 gcgttgctag atctccattg tctattaatt cgacgccaaa agatgctgac atattttcca
     1381 gttggttgct tttgaaaaat ctaacagcac agattgatgg tcctactttg cgtacactgt
     1441 gtatgcagca tgggcccctt gtcagctttc acccgtattt gaaccaagga attgccttat
     1501 gtaaatatac tactcgtgag gaggcgaaca aggcgcaaat ggcgttaaac aactgtgtcc
     1561 tcgccaacac cacaatattt gctgaatctc ccagcgagaa cgaggtgcaa agcattatgc
     1621 agcacttatc taggggtcga ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg
     1681 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     1741 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     1801 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     1861 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     1921 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1981 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2041 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2101 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2161 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2221 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2281 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2341 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2401 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2461 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2521 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2581 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     2641 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2701 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2761 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2821 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2881 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2941 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3001 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3241 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3721 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3781 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3841 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3901 gcggtatttc acaccgcata aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     3961 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4021 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4081 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4141 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4201 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4261 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4321 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4381 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4441 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     4501 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     4561 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     4621 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     4681 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     4741 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     4801 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     4861 caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     4921 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     4981 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5041 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5101 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5161 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5221 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5281 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5341 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     5401 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     5461 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     5521 aacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     5581 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     5641 tgatggcgtt ggaatt
//
