LOCUS       sequence-332119-        5945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial experssion of a GST-tagged MEG-3 C-terminus with mutations
            in the HMG-like motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5945)
  AUTHORS   Schmidt H, Putnam A, Rasoloson D, Seydoux G
  TITLE     Coordination of RNA and protein condensation by the P granule 
            protein MEG-3
  JOURNAL   bioRxiv 2020.10.15.340570
REFERENCE   2  (bases 1 to 5945)
  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"; journalName: "bioRxiv 
            2020.10.15.340570"
COMMENT     Sequence Label: pHS33
FEATURES             Location/Qualifiers
     source          1..5945
                     /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(1995..2017)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2162..2180)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2248..2352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2353..2421,2422..3213)
                     /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(2571..2590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3384..3972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3873..3892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4126..4143
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4216..4293
                     /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             4294..5376
                     /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(4313..4332)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5389..5410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5425..5430,5431..5448,5449..5455)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5463..5479
                     /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     5487..5503
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5487..5503
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5499..5945,1..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(5519..5536)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5519..5535)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5528..5550)
                     /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 ccggaattcc
      961 cgggtcgaca catgtcgtca atcatcccaa ctccaaaggc tttgatcgga gaaagatgtc
     1021 tgacatcatc cagcaagtct tcaaagttga acaagtcact tggcgtggtt gattctaaag
     1081 ctacaaaatc accaatgtat tccgtcacag tttctggaaa agaaacagct tcaggaaagc
     1141 gaattgctca aaagttgact ccaaaggttg tggcgctgga aagctcttac attaccggaa
     1201 ttccagtttc cacggattgt aatggttgcc caactccgaa gagaagtgga atcaattgtg
     1261 aaattcgtgc agctgaagta tacaatcaag ctgggaaatg gcccttcgaa atcaccagtg
     1321 atccggctcc acttccttgt gaatcagccg atcggatcga atatccttct caagattgca
     1381 ctcaagatcc agcttcaacc tcgccaccac ctcgcatttc tgaaagcttg acagcattcc
     1441 tggaagctca gcaagacgcc aacgacgcta ttgatactaa cgccaaagaa aagacacaac
     1501 tcctgaaagt gaatttggct attcacggga tgtcacctga aagatggctg tacttgaatt
     1561 atttttgcac cgagacaatt ccaagattgg atggtcctta tgccgatgat ccaagagttc
     1621 cacctgtgcg aaatatgttc agaaaatggt tcctccgttt tgctgaagcc tgcttaggaa
     1681 atcctcacca gcttgcagtt atgcaagaga ttgctgccac gtttgttcag gctcggctgg
     1741 acgatacgag cagctccaca gactccacca atatgctcta catgttgtgg aaggagtgta
     1801 tcggacagaa gaatatcatc gcaattgctg atgcgtgttt acttgcacat cttcggaaat
     1861 cggatcctat caaatacttg aatgtaaaac gggattggct ggaatctatt tttgacccac
     1921 ccagagatca atgagcggcc gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt
     1981 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     2041 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     2101 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag
     2161 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     2221 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2281 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2341 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2401 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2461 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2521 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2581 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2641 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2701 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2761 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2821 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2881 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2941 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3001 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3061 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3121 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3181 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3241 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3301 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3361 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3421 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3481 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3541 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3601 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3661 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3721 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3781 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3841 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3901 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3961 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4021 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4081 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4141 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4201 caccgcataa attccgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
     4261 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     4321 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     4381 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     4441 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     4501 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     4561 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     4621 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     4681 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     4741 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     4801 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     4861 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     4921 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     4981 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     5041 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     5101 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtc aaccaccatc
     5161 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     5221 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     5281 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5341 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5401 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5461 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac
     5521 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     5581 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     5641 cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag
     5701 aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc
     5761 cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgta acctatccca
     5821 ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat
     5881 ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg
     5941 gaatt
//
