LOCUS       sequence-289319-        6197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmGW182_862-1384-del1004-1115.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6197)
  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 6197)
  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_862-1384-del1004-1115_L
FEATURES             Location/Qualifiers
     source          1..6197
                     /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(2247..2269)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2414..2432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2500..2604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2605..2673,2674..3465)
                     /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(2823..2842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3636..4224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4125..4144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4378..4395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4468..4545
                     /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             4546..5628
                     /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(4565..4584)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5641..5662
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5677..5682,5683..5700,5701..5707)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5715..5731
                     /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     5739..5755
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5739..5755
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5751..6197,1..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(5771..5788)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5771..5787)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5780..5802)
                     /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 agcacttacc acaaactcct tcctctacaa gctctagtgg aactagtggt ggcaacgtcg
     1681 gaggcgtcgg cacttcagcc aataatgcaa acagtggttc tgcagcttgt ctgtccggaa
     1741 acaatagcgg caacggaaac ggcagcgcga gcggcgccgg cagcggcaac aatggcaaca
     1801 gtagctgcaa caacagtgcc gccggggggg gcagcagcag caacaacacg attaccactg
     1861 tagcaaattc gaatcttgtt ggttctagtg gctctgtctc aaattcctct ggcgttactg
     1921 ctaactctag tactgtttct gtagttagtt gtacagcgag tgggaattcc ataaatgggg
     1981 caggtactgc aaacagttct ggttcaaaga gtagtgcaaa caatttagct agcggccagt
     2041 ctagcgcttc taacttaact aatagcacca attcaacatg gcgacaaact agccaaaacc
     2101 aagctcttca aagtcaaagc aggccatcag gcagagaagc tgactttgat tatatatctc
     2161 tcgtttattc cattgttgat gattaagcgg ccgcatcgtg actgactgac gatctgcctc
     2221 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     2281 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     2341 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtataattct
     2401 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2461 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2521 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2581 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2641 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2701 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2761 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2821 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     2881 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2941 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3001 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3061 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3121 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     3181 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3241 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3301 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3361 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3421 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3481 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3541 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3601 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3661 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3721 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3781 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3841 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3901 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3961 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4021 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4081 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4141 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4201 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4261 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4321 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4381 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4441 tgcggtattt cacaccgcat aaattccgac accatcgaat ggtgcaaaac ctttcgcggt
     4501 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     4561 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     4621 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     4681 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     4741 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     4801 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     4861 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     4921 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     4981 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     5041 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     5101 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     5161 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     5221 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     5281 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     5341 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     5401 tcaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     5461 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     5521 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5581 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5641 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     5701 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     5761 ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     5821 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     5881 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt
     5941 ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata
     6001 ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg
     6061 taacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt
     6121 actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt
     6181 ttgatggcgt tggaatt
//
