LOCUS       sequence-255788-        7186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for (SIM)5 client to assess SUMO-SIM 
            scaffolds..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7186)
  AUTHORS   Banani SF, Rice AM, Peeples WB, Lin Y, Jain S, Parker R, Rosen MK
  TITLE     Compositional Control of Phase-Separated Cellular Bodies.
  JOURNAL   Cell. 2016 Jul 28;166(3):651-663. doi: 10.1016/j.cell.2016.06.010. 
            Epub 2016 Jun 30.
  PUBMED    27374333
REFERENCE   2  (bases 1 to 7186)
  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.2016.06.010"; journalName: "Cell"; date: 
            "2016-07-28- 28"; volume: "166"; issue: "3"; pages: "651-663"
COMMENT     Sequence Label: (SIM)5
FEATURES             Location/Qualifiers
     source          1..7186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..48
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     132..151
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        132..150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    151..175
                     /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             190..212
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             228..257
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             261..1361
                     /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      1218..1227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1335..1358
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1410..1421
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1428..1448
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1986..2006
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2049..2066
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2119..2137)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2133..2180
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2217..2672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2304..2323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2514..2535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2698..2802
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2803..2871,2872..3663)
                     /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(3021..3040)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3834..4422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4323..4342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4576..4593
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4608..4747
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4673..4692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4833..4855
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4849..5040)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5849..6931)
                     /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     6893..6912
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6932..7009)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
       61 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
      121 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
      181 agaaataatt ttgtttaact ttaagaagga gatataccat gtctggccat caccatcacc
      241 atcaccatca ccatcacggc atgaaaatcg aagaaggtaa actggtaatc tggattaacg
      301 gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa
      361 ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa
      421 ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat
      481 ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta
      541 cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt
      601 tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc
      661 cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag
      721 aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa
      781 acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga
      841 ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg
      901 cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt
      961 ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc
     1021 aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca
     1081 aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg
     1141 ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga
     1201 aagatccacg tattgccgcc accatggaaa acgcccagaa aggtgaaatc atgccgaaca
     1261 tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg
     1321 gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc tcgaacaaca
     1381 acaacaataa caataacaac aacctcggga tcgagggaag gatttcagaa aacctgtact
     1441 tccagggatc cggtggctct tggggtggga gcaaagtaga tgtgatcgac ttgaccatcg
     1501 aaagcagcag cgacgaagaa gaagatccgc ctgccaaacg cggcggtagc ggtggctctg
     1561 gcgggagcgg cggctcgaaa gttgatgtca ttgaccttac aattgaatct tcgagcgatg
     1621 aagaggaaga tcctccagcg aaacgtggcg gctcgggagg ttcgggcggc agcggcggaa
     1681 gcaaagtcga tgtaattgac ttaacgatcg aatctagcag cgatgaagaa gaagatccac
     1741 cggctaaacg tgggggctca gggggttctg gcggttcagg cggcagtaaa gttgacgtga
     1801 ttgacctcac catcgaaagc agtagcgacg aggaagaaga tccaccagca aaacgcggag
     1861 ggagcggggg cagcggcggc agcggcggat ctaaagtgga cgttatcgat ctgacgatcg
     1921 aaagcagtag cgatgaggaa gaggacccgc cggcgaaacg gggcggctcg tgcgggggct
     1981 cactcgagaa cctgtatttt cagggccgta aacgtaaacg taaacgtaaa cgtaaaggct
     2041 gagtcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     2101 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2161 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2221 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2281 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2341 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2401 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2461 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2521 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2581 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2641 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2701 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2761 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2821 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2881 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2941 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3001 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3061 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3121 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3181 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3241 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3301 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     3361 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3421 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3481 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3541 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3601 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3661 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3721 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3781 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3841 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3901 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3961 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4021 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4081 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4141 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4201 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4261 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4321 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4381 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4441 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4501 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4561 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4621 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4681 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4741 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4801 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4861 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4921 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4981 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5041 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5101 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5161 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5221 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5281 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5341 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5401 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5461 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5521 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5581 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5641 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5701 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5761 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5821 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5881 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5941 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6001 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6061 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6121 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6181 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6241 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6301 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6361 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6421 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6481 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6541 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6601 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6661 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6721 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6781 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6841 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6901 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6961 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7021 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7081 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7141 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcc
//
