LOCUS       sequence-341027-        7909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression for HIS purification of aa1-698 of MEG-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7909)
  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 7909)
  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: pDC32
FEATURES             Location/Qualifiers
     source          1..7909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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)."
     RBS             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1480..1497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1507..1524
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4154..4171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4224..4242)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4238..4285
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4322..4777
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4409..4428)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4619..4640
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4869..5684)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4869..5684)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5592..5611
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5676..5695
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5806..6394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6295..6314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6548..6565
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6580..6719
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6645..6664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6805..6827
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6821..7012)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7821..7909,1..994))
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
     1501 agcggcctgg tgccgcgcgg cagccatatg agttcctcaa aaccttaccc aagcggtctg
     1561 ccaaactcga gaagaaaaag aggaggtcgc cgctcaagta gtcgtagcaa tcaggaaagt
     1621 gcatccaaca acatggaaca ccaaatcact cttgacgaat tgttcaatcc gatcgcaaaa
     1681 caagacagtg ctcagtcaac aagccgtgaa tatggtgcaa aatcggggat atctcatcac
     1741 ggatccgtat cattcaacgg gaatacattc atgaatggcc aacagctgaa tcactcgatg
     1801 actcgacatg gtcgagtatt caatcagtcg atgcatgcag ctcaaggaaa cggcagcaac
     1861 gcctttaaca gtattcctcc tacggccccc gtcttctctg cagacttccg ccgcaacttg
     1921 caaacacgca atagctccag ctggtatgaa cgaagattcc cagtatccac agatcaggac
     1981 gacgttcagc aatcaaacac aagacgtagt agaagtcgtc agaatggaca acatggccta
     2041 agcttctcgg atggttcgaa caattatggt cacgctggga ataagtcatt cagtgtgagt
     2101 tccgttcccg ttgggttcca gaagcaagaa aataattcta agaaactgcg acaaaccaat
     2161 gttcaccagc aatgtcttgg aaataaaagc ttcaacgctc aagctggagt tcatggacac
     2221 gcttttaaaa aaggccataa ggacaataaa aatgcatctg gaaaggaggt tatcaattct
     2281 agtcttgtcc aaaagcacga tgctattaag tcgcggaatt tgaaccagag cttctctgga
     2341 tttcctacac atgaaacctc atcgatgaaa aatcaacaac agaaatcaag aaacgacaga
     2401 aaaaaatcac gtggtagcag caacttccag gatcgtactt acttcaacac aaatgacgat
     2461 gaattaactg acgatgtgtt catagacgat tccatggatg ctgcccgtgg tcggagatct
     2521 cgatcagtca ctaagaaact tcaacaatcg acttattcaa agcagaatgc aggcagtaaa
     2581 caattgactg aaaaatgcaa atcttctgaa gaagcggcta aacgtaacct ggtatccaat
     2641 gtattcagca aggacggcac tgaactctcc attgaacagt tattggaaat tgtcagcatg
     2701 aagatcggac aacagattca cttgccttcc agctctcatg gagaatgcag caatttgaat
     2761 cggacattac cagcaagtga tctaaattgc tcaattggtg aagactttga ctccagtttt
     2821 gtggatgcta acaaccaaac cctaccagtg agtcttccca agaaaacttc actgtcgatc
     2881 aagcgccgtg gaagttctcg tagtgcctcc cgtctggcca gtcttgatgt gacactggaa
     2941 actgttgaag aagacgagga acccacgcca agtccacagc cgagttctcc tccaaagatc
     3001 tcaagaagaa agtggacagg aacttttgat gccaacgttg aggaaatgag acgtttactt
     3061 catggggacc ctgagatgcc gaaatctgca aatcgtgctt cctcaagcaa ggatcagatc
     3121 aatcggaaca atgtggatgt aaagagaaca ccttcctcgt caatcatccc aactccaaag
     3181 gctttgatcg gagaaagatg tctgacatca tccagcaagt cttcaaagtt gaacaagtca
     3241 cttggcgtgg ttgattctaa agctacaaaa tcaccaatgt attccgtcac agtttctgga
     3301 aaagaaacag cttcaggaaa gcgaattgct caaaagttga ctccaaaggt tgtggcgctg
     3361 gaaagctctt acattaccgg aattccagtt tccacggatt gtaatggttg cccaactccg
     3421 aagagaagtg gaatcaattg tgaaattcgt gcagctgaag tatacaatca agctgggaaa
     3481 tggcccttcg aaatcaccag tgatccggct ccacttcctt gtgaatcagc cgatcggatc
     3541 gaatatcctt ctcaagattg cactcaagat ccagcttcaa cctcgccacc acctcgcatt
     3601 tctgaaagct tgacagcatt ctgatgagaa gctcagcaag actttaacga ctacattgat
     3661 actaactaca aagaaaagac acaactcctg aaagtgaatt tgaatattca cgggatgtca
     3721 cctgaaagat ggctgtactt gaattatttt tgcaccgaga caattccaag attggatggt
     3781 ccttatgccg atgatccaag agttccacct gtgcgaaata tgttcagaaa atggttcctc
     3841 cgttttgctg aagcctgctt aggaaatcct caccagcttg cagttatgca agagattgct
     3901 gccacgtttg ttcaggctcg gctggacgat acgagcagct ccacagactc caccaatatg
     3961 ctctacatgt tgtggaagga gtgtatcgga cagaagaata tcatcgcaat tgctgatgcg
     4021 tgtttacttg cacatcttcg gaaatcggat cctatcaaat acttgaatgt aaaacgggat
     4081 tggctggaat ctatttttga cccacccaga gatcaatgag agctccgtcg acaagcttgc
     4141 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     4201 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     4261 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     4321 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4381 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4441 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4501 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4561 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4621 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4681 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4741 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     4801 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4861 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4921 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4981 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     5041 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     5101 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     5161 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     5221 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     5281 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     5341 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     5401 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     5461 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     5521 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     5581 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     5641 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     5701 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     5761 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5821 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5941 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6181 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6421 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6481 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6541 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     6601 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     6661 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     6721 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     6781 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     6841 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     6901 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     6961 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     7021 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     7081 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     7141 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     7201 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     7261 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     7321 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     7381 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     7441 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     7501 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     7561 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     7621 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     7681 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     7741 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     7801 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     7861 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattgg
//
