LOCUS       sequence-232143-        6838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector for 6 sim.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6838)
  AUTHORS   Delarue M, Brittingham GP, Pfeffer S, Surovtsev IV, Pinglay S, 
            Kennedy KJ, Schaffer M, Gutierrez JI, Sang D, Poterewicz G, Chung 
            JK, Plitzko JM, Groves JT, Jacobs-Wagner C, Engel BD, Holt LJ
  TITLE     mTORC1 Controls Phase Separation and the Biophysical Properties of 
            the Cytoplasm by Tuning Crowding.
  JOURNAL   Cell. 2018 Jul 12;174(2):338-349.e20. doi: 
            10.1016/j.cell.2018.05.042. Epub 2018 Jun 21.
  PUBMED    29937223
REFERENCE   2  (bases 1 to 6838)
  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: 
            "10.1016/j.cell.2018.05.042"; journalName: "Cell"; date: 
            "2018-07-12- 12"; volume: "174"; issue: "2"; pages: "338-349.e20"
COMMENT     Sequence Label: pT7-HISx6-SIMx6
FEATURES             Location/Qualifiers
     source          1..6838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(216..1298)
                     /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"
     primer_bind     1260..1279
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1299..1376)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1582..1601
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1689..1708
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1689..1707
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1708..1732
                     /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             1749..1771
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1797..1814
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1815..1835
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1854..1856,1857..2177)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             3387..3404
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3457..3475)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3471..3518
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3555..4010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3642..3661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3852..3873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4102..4917)
                     /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(4102..4917)
                     /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     4825..4844
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4909..4928
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5039..5627
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5528..5547
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5781..5798
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5813..5952
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5878..5897)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6038..6060
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6054..6245)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
       61 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
      121 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
      181 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      241 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      301 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      361 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      421 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      481 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      541 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      601 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      661 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      721 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      781 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      841 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
      901 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
      961 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1021 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1081 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1141 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1201 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1261 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1321 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1381 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1441 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1501 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     1561 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1621 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     1681 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt ccccttaatt
     1741 aaaataattt tgtttaactt taagaaggag atatacataa gcttatgggc agcagccatc
     1801 accatcatca ccacgagaat ttgtattttc agggtagcca agatccgtgt acaatgggag
     1861 tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc ggccagacct
     1921 gcgtggtgca ctacaccggg atgcttgaag atggaaagaa atttgattcc tcccgggaca
     1981 gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc tgggaagaag
     2041 gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca gattatgcct
     2101 atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc ttcgatgtgg
     2161 agcttctaaa actggaaggt tcatccggat cttcagggag ttctggttca agcactagta
     2221 aggttgatgt aatagatttg accatagaat ctagttctga cgaggaagaa gatcctcctg
     2281 caaagaggga attcgtcgta gggggctccg gcggaagcgg ggggtcggga ggttccaagg
     2341 ttgatgtaat agatttgacc atagaatcta gttctgacga ggaagaagat cctcctgcaa
     2401 agagggttgt tggcggaagc ggcgggagcg gtggtagcgg gggttctaag gttgatgtaa
     2461 tagatttgac catagaatct agttctgacg aggaagaaga tcctcctgca aagagggtag
     2521 tgggtggctc cggtggctct ggtgggtctg gtggctcgaa ggttgatgta atagatttga
     2581 ccatagaatc tagttctgac gaggaagaag atcctcctgc aaagagggtg gtgggaggga
     2641 gcggcggtag cgggggatca ggtggtagta aggttgatgt aatagatttg accatagaat
     2701 ctagttctga cgaggaagaa gatcctcctg caaagagggt cgtaggttca tccggatctt
     2761 cagggagttc tggttcaagc aaggttgatg taatagattt gaccatagaa tctagttctg
     2821 acgaggaaga agatcctcct gcaaagaggg aattcgtggt gggcggctcc gggggaagcg
     2881 gggggtccgg aggtagtaga tcttaagtcg taggatcttc aggatcaagc ggttcatctg
     2941 gtagttccgg atccaaggtt gatgtaatag atttgaccat agaatctagt tctgacgagg
     3001 aagaagatcc tcctgcaaag agggttgttg gatcttcagg atcaagcggt tcatctggta
     3061 gttccaaggt tgatgtaata gatttgacca tagaatctag ttctgacgag gaagaagatc
     3121 ctcctgcaaa gagggttgtt ggttcctctg gaagctcagg atctagtggg tcatcaaagg
     3181 ttgatgtaat agatttgacc atagaatcta gttctgacga ggaagaagat cctcctgcaa
     3241 agagggttgt tggttcatca ggttcatccg gatctagcgg gtcaagtaag gttgatgtaa
     3301 tagatttgac catagaatct agttctgacg aggaagaaga tcctcctgca aagaggtcta
     3361 gataaaccgg tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     3421 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     3481 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     3541 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3601 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3661 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3721 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3781 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3841 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3901 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3961 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     4021 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4081 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     4141 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     4201 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     4261 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     4321 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     4381 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     4441 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     4501 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     4561 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     4621 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     4681 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     4741 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     4801 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     4861 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     4921 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     4981 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5041 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5101 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5161 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     5221 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5281 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5341 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5401 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5461 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5521 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5581 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5641 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5701 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5761 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     5821 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     5881 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     5941 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     6001 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     6061 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     6121 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     6181 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     6241 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     6301 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     6361 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     6421 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     6481 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     6541 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     6601 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     6661 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     6721 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     6781 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcg
//
