LOCUS       sequence-289315-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of DmGW182_862-1199.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  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 5992)
  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_L
FEATURES             Location/Qualifiers
     source          1..5992
                     /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(2042..2064)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2209..2227)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2295..2399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2400..2468,2469..3260)
                     /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(2618..2637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3431..4019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3920..3939
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4173..4190
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4263..4340
                     /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             4341..5423
                     /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(4360..4379)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5436..5457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5472..5477,5478..5495,5496..5502)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5510..5526
                     /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     5534..5550
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5534..5550
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5546..5992,1..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(5566..5583)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5566..5582)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5575..5597)
                     /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 cttatctagg ggtcgactcg
     1981 agcggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa
     2041 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     2101 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     2161 cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac
     2221 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2281 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2341 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2401 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2461 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2521 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2581 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2641 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2701 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2761 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2821 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2881 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2941 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3001 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3061 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3121 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3181 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3241 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3301 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3361 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3421 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3481 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3541 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3601 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3661 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3721 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3781 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3841 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3901 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3961 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4021 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4081 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4141 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4201 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt
     4261 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     4321 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     4381 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     4441 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     4501 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     4561 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     4621 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     4681 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     4741 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     4801 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     4861 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     4921 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     4981 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     5041 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     5101 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     5161 acgataccga agacagctca tgttatatcc cgccgtcaac caccatcaaa caggattttc
     5221 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     5281 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     5341 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     5401 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     5461 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     5521 taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt
     5581 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     5641 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     5701 gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
     5761 aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca
     5821 gatgcacggt tacgatgcgc ccatctacac caacgtaacc tatcccatta cggtcaatcc
     5881 gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga
     5941 aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa tt
//
