LOCUS       sequence-255787-        6544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for eGFP-(SIM)3 client to assess 
            SUMO-SIM scaffolds..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6544)
  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 6544)
  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: GFP-(SIM)3
FEATURES             Location/Qualifiers
     source          1..6544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             75..104
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             108..128
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(132..134,135..137,138..848)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(177..198)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(438..457)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     785..806
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1194..1214
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1257..1274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1327..1345)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1341..1388
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1425..1880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1512..1531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1722..1743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1906..2010
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2011..2079,2080..2871)
                     /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(2229..2248)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3042..3630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3531..3550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3784..3801
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3816..3955
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3881..3900)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4041..4063
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4057..4248)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5057..6139)
                     /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     6101..6120
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6140..6217)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6423..6442
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6526..6544,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6526..6544
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggccatcac catcaccatc accatcacca tcacggcgaa aacctatact
      121 tccagggatc catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      181 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
      241 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
      301 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
      361 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
      421 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
      481 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
      541 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
      601 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
      661 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
      721 ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg
      781 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
      841 tgtacaagtc cggaggtagt ggcggtagcg gtggctcttg gggtgggagc aaagtagatg
      901 tgatcgactt gaccatcgaa agcagcagcg acgaagaaga agatccgcct gccaaacgcg
      961 gcggtagcgg tggctctggc gggagcggcg gctcgaaagt tgatgtcatt gaccttacaa
     1021 ttgaatcttc gagcgatgaa gaggaagatc ctccagcgaa acgtggcggc tcgggaggtt
     1081 cgggcggcag cggcggaagc aaagtcgatg taattgactt aacgatcgaa tctagcagcg
     1141 atgaagaaga agatccaccg gctaaacgtg ggggctcatg cgggggttct ctcgagaacc
     1201 tgtattttca gggccgtaaa cgtaaacgta aacgtaaacg taaaggctga gtcgagcacc
     1261 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1321 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1381 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1441 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1501 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1561 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1621 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1681 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1741 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1801 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1861 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1921 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1981 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2041 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2101 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2161 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2221 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     2281 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2341 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2401 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2461 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2521 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     2581 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2641 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2701 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     2761 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2821 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2881 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2941 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3001 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3061 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3121 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3181 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3241 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3301 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3361 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3421 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3481 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3541 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3601 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3661 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3721 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3781 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3841 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3901 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3961 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4021 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4081 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4141 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4201 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4261 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4321 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4381 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4441 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4501 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4561 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4621 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     4681 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     4741 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4801 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4861 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4921 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4981 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5041 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5161 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5221 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5281 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     5341 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5401 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5461 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5521 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5581 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5641 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5701 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5761 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5821 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5881 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5941 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6001 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6061 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6121 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6181 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6241 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6301 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6361 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6421 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6481 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     6541 tagg
//
