LOCUS       sequence-459203-        7711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of polySIM protein under T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7711)
  AUTHORS   Kim J, Qin S, Zhou HX, Rosen MK
  TITLE     Surface Charge Can Modulate Phase Separation of Multidomain 
            Proteins.
  JOURNAL   J Am Chem Soc. 2024 Feb 7;146(5):3383-3395. doi: 
            10.1021/jacs.3c12789. Epub 2024 Jan 23.
  PUBMED    38262618
REFERENCE   2  (bases 1 to 7711)
  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: 
            "10.1021/jacs.3c12789"; journalName: "J Am Chem Soc"; date: 
            "2024-02-7- 7"; volume: "146"; issue: "5"; pages: "3383-3395"
COMMENT     Sequence Label: polySIM
FEATURES             Location/Qualifiers
     source          1..7711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(24..1106)
                     /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     1068..1087
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1107..1184)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1390..1409
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1493..1512
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1493..1511
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1512..1536
                     /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             1551..1573
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1589..1618
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1622..2722
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2579..2588
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2696..2719
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2771..2782
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2789..2809
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3872..3892
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3935..3952
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4005..4023)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4019..4066
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4103..4558
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4190..4209)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4400..4421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4584..4688
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4689..4757,4758..5549)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4907..4926)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5720..6308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6209..6228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6462..6479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6494..6633
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6559..6578)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6719..6741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6735..6926)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
       61 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
      121 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
      181 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
      241 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
      301 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
      361 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
      421 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
      481 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
      541 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
      601 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
      661 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
      721 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
      781 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
      841 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
      901 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
      961 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     1021 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     1081 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     1141 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     1201 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     1261 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     1321 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     1381 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     1441 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     1501 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
     1561 tttaagaagg agatatacca tgtctggcca tcaccatcac catcaccatc accatcacgg
     1621 catgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
     1681 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
     1741 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
     1801 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
     1861 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
     1921 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
     1981 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
     2041 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
     2101 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
     2161 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
     2221 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
     2281 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
     2341 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
     2401 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     2461 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     2521 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     2581 caccatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     2641 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     2701 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     2761 caacctcggg atcgagggaa ggatttcaga aaacctgtac ttccagggat ccggtggctc
     2821 ttggggtggg agcaaagtag atgtgatcga cttgaccatc gaaagcagca gcgacgaaga
     2881 agaagatccg cctgccaaac gcggcggtag cggtggctct ggcgggagcg gcggctcgaa
     2941 agttgatgtc attgacctta caattgaatc ttcgagcgat gaagaggaag atcctccagc
     3001 gaaacgtggc ggctcgggag gttcgggcgg cagcggcgga agcaaagtcg atgtaattga
     3061 cttaacgatc gaatctagca gcgatgaaga agaagatcca ccggctaaac gtgggggctc
     3121 agggggttct ggcggttcag gcggcagtaa agttgacgtg attgacctca ccatcgaaag
     3181 cagtagcgac gaggaagaag atccaccagc aaaacgcgga gggagcgggg gcagcggcgg
     3241 cagcggcgga tctaaagtgg acgttatcga tctgacgatc gaaagcagta gcgatgagga
     3301 agaggacccg ccggcgaaac ggggcggctc ggggggatca ggtggctctg gtgggagcaa
     3361 agtagatgtg atcgacttga ccatcgaaag cagcagcgac gaagaagaag atccgcctgc
     3421 caaacgcggc ggtagcggtg gctctggcgg gagcggcggc tcgaaagttg atgtcattga
     3481 ccttacaatt gaatcttcga gcgatgaaga ggaagatcct ccagcgaaac gtggcggctc
     3541 gggaggttcg ggcggcagcg gcggaagcaa agtcgatgta attgacttaa cgatcgaatc
     3601 tagcagcgat gaagaagaag atccaccggc taaacgtggg ggctcagggg gttctggcgg
     3661 ttcaggcggc agtaaagttg acgtgattga cctcaccatc gaaagcagta gcgacgagga
     3721 agaagatcca ccagcaaaac gcggagggag cgggggcagc ggcggcagcg gcggatctaa
     3781 agtggacgtt atcgatctga cgatcgaaag cagtagcgat gaggaagagg acccgccggc
     3841 gaaacggggc ggctcgtgcg ggggctcact cgagaacctg tattttcagg gccgtaaacg
     3901 taaacgtaaa cgtaaacgta aaggctgagt cgagcaccac caccaccacc actgagatcc
     3961 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     4021 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     4081 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4141 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4201 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4261 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     4321 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4381 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4441 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4501 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     4561 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4621 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4681 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4741 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4801 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4861 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4921 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4981 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5041 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5101 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5161 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5221 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5281 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5341 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5401 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5461 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5521 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5581 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5641 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5701 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5761 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5821 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     5881 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5941 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6001 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6061 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6121 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6181 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6241 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6301 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6361 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6421 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6481 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     6541 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     6601 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     6661 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     6721 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     6781 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     6841 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     6901 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     6961 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     7021 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     7081 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     7141 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     7201 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     7261 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     7321 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     7381 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     7441 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     7501 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     7561 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     7621 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     7681 ggtcgagatc ccggtgccta atgagtgagc t
//
