LOCUS       sequence-289317-        6533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmGW182_862-1384.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6533)
  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 6533)
  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_L
FEATURES             Location/Qualifiers
     source          1..6533
                     /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(2583..2605)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2750..2768)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2836..2940
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2941..3009,3010..3801)
                     /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(3159..3178)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3972..4560
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4461..4480
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4714..4731
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4804..4881
                     /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             4882..5964
                     /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(4901..4920)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5977..5998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6013..6018,6019..6036,6037..6043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6051..6067
                     /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     6075..6091
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6075..6091
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6087..6533,1..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(6107..6124)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6107..6123)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6116..6138)
                     /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 atatttgcca
     1381 ataccggtaa aaattcaccg actgatttac cgccactaag tttatcgtcg tctacatgga
     1441 gttttaatcc aaaccaaaat tatccgagtc acagttggtc tgacaatagt caacaatgta
     1501 ccgccacttc ggagctttgg acaagcccgc taaataaatc atcgtctcga ggtcccccgc
     1561 caggattgac tgccaattca aataaatctg caaatagtaa tgcgtcaacg ccaacaacta
     1621 ttaccggagg tgcgaatgga tggttacagc ctcgaagtgg cggtgttcaa accacaaaca
     1681 ctaattggac aggtggtaac accacttggg gctccagttg gttgcttttg aaaaatctaa
     1741 cagcacagat tgatggtcct actttgcgta cactgtgtat gcagcatggg ccccttgtca
     1801 gctttcaccc gtatttgaac caaggaattg ccttatgtaa atatactact cgtgaggagg
     1861 cgaacaaggc gcaaatggcg ttaaacaact gtgtcctcgc caacaccaca atatttgctg
     1921 aatctcccag cgagaacgag gtgcaaagca ttatgcagca cttaccacaa actccttcct
     1981 ctacaagctc tagtggaact agtggtggca acgtcggagg cgtcggcact tcagccaata
     2041 atgcaaacag tggttctgca gcttgtctgt ccggaaacaa tagcggcaac ggaaacggca
     2101 gcgcgagcgg cgccggcagc ggcaacaatg gcaacagtag ctgcaacaac agtgccgccg
     2161 gggggggcag cagcagcaac aacacgatta ccactgtagc aaattcgaat cttgttggtt
     2221 ctagtggctc tgtctcaaat tcctctggcg ttactgctaa ctctagtact gtttctgtag
     2281 ttagttgtac agcgagtggg aattccataa atggggcagg tactgcaaac agttctggtt
     2341 caaagagtag tgcaaacaat ttagctagcg gccagtctag cgcttctaac ttaactaata
     2401 gcaccaattc aacatggcga caaactagcc aaaaccaagc tcttcaaagt caaagcaggc
     2461 catcaggcag agaagctgac tttgattata tatctctcgt ttattccatt gttgatgatt
     2521 aagcggccgc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa
     2581 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     2641 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     2701 acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg cctcgtgata
     2761 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     2821 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2881 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2941 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3001 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3061 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3121 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3181 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3241 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3301 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3361 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3421 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3481 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3541 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3601 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3661 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3721 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3781 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3841 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3901 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3961 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4021 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4081 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4141 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4201 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4261 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4321 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4381 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4441 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4501 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4561 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4621 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4681 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4741 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat
     4801 tccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     4861 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     4921 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     4981 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     5041 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     5101 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     5161 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     5221 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     5281 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     5341 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     5401 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     5461 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     5521 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     5581 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     5641 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     5701 gacgataccg aagacagctc atgttatatc ccgccgtcaa ccaccatcaa acaggatttt
     5761 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     5821 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     5881 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     5941 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     6001 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     6061 ataacaattt cacacaggaa acagctatga ccatgattac ggattcactg gccgtcgttt
     6121 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     6181 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     6241 tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg
     6301 aaagctggct ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc
     6361 agatgcacgg ttacgatgcg cccatctaca ccaacgtaac ctatcccatt acggtcaatc
     6421 cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg
     6481 aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttgga att
//
