LOCUS       sequence-485116-        6308 bp    DNA     circular SYN 02-SEP-2025
DEFINITION  Expresses the MBP-Streptavidin-RGG fusion protein in E Coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6308)
  AUTHORS   Kelley FM, Ani A, Pinlac EG, Linders B, Favetta B, Barai M, Ma Y, 
            Singh A, Dignon GL, Gu Y, Schuster BS
  TITLE     Controlled and orthogonal partitioning of large particles into 
            biomolecular condensates.
  JOURNAL   Nat Commun. 2025 Apr 14;16(1):3521. doi: 10.1038/s41467-025-58900-5.
  PUBMED    40229261
REFERENCE   2  (bases 1 to 6308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-04-14"; volume: "16(1):3521. doi"; pages: " 
            10.1038/s41467-025-58900-5"
COMMENT     Sequence Label: MBP-Streptavidin-RGG
FEATURES             Location/Qualifiers
     source          1..6308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     861..880
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(900..977)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1183..1202
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1286..1305
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1286..1304
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1305..1329
                     /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             1344..1366
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1377..1394
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      1391..1400
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1407..2507
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2481..2504
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2556..2576
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2961..2978
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3489..3506
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3559..3577)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3573..3620
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3657..4112
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3744..3763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3954..3975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4227..5042)
                     /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(4227..5042)
                     /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     4950..4969
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5034..5053
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5195..5783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5684..5703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5937..5954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(6125..6308,1..899))
                     /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 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
       61 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      121 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      181 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      241 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      301 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      361 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      421 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      481 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      541 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      601 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      661 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      721 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      781 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      841 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      901 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
      961 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1021 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1081 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1141 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1201 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1261 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1321 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgcacc
     1381 atcaccatca ccatggcggc ggatctatga aaatcgaaga aggtaaactg gtaatctgga
     1441 ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata
     1501 ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg
     1561 cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg
     1621 ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc
     1681 cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg
     1741 aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag
     1801 agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc
     1861 tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt
     1921 atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg
     1981 gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact
     2041 ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg
     2101 catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca
     2161 agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc
     2221 cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg
     2281 aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt
     2341 tggtgaaaga tccgcgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc
     2401 cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg
     2461 ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagaccaat tcgagctcga
     2521 acaacaacaa caataacaat aacaacaacc tcggagagaa cctgtatttc cagggcatgg
     2581 ctgaagctgg tatcaccggc acctggtaca accagctggg atccaccttc atcgttaccg
     2641 ctggtgctga cggtgctctg accggtacct acgaatccgc tgttggtaac gctgaatcta
     2701 gatacgttct gaccggtcgt tacgactccg ctccggctac cgacggttcc ggaaccgctc
     2761 tgggttggac cgttgcttgg aaaaacaact accgtaacgc tcactccgct accacctggt
     2821 ctggccagta cgttggtggt gctgaagctc gtatcaacac ccagtggttg ttgacctccg
     2881 gcaccaccga agccaacgcg tggaaatcca ccctggttgg tcacgacacc ttcaccaaag
     2941 ttaaaccgtc cgctgcttcc ttagtacctc gcgggtcaat ggaatccaat caatccaata
     3001 acggtggctc gggtaatgcc gctctgaatc gtggtggccg ctacgtgccg ccgcatctgc
     3061 gtggtggcga tggtggtgca gcagcagctg catctgctgg cggtgatgac cgtcgcggcg
     3121 gtgcgggcgg tggcggttat cgtcgcggcg gtggcaatag tggtggcggt ggcggtggcg
     3181 gttatgatcg tggttacaat gacaaccgtg atgaccgcga taaccgtggc ggttccggcg
     3241 gttacggtcg tgatcgtaat tatgaagacc gtggttacaa cggcggtggc ggtggcggtg
     3301 gcaatcgtgg ctataacaat aaccgtggtg gcggtggcgg tggctacaat cgccaggatc
     3361 gtggtgacgg tggcagctct aacttttcgc gcggtggcta taataaccgt gatgaaggca
     3421 gcgacaaccg cggtagtggc cgttcctaca ataacgatcg tcgcgacaat ggtggcgatg
     3481 gcctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     3541 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     3601 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     3661 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3721 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3781 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3841 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3901 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3961 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4021 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4081 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg tacccaggtg
     4141 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4201 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     4261 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     4321 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     4381 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     4441 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     4501 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     4561 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     4621 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     4681 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     4741 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     4801 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     4861 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     4921 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     4981 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     5041 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     5101 cccttaacgt gacctaggtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5161 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5221 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5281 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5341 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5401 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5461 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5521 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5581 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5641 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5701 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5761 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5821 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5881 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5941 agtcagtgag cgaggaagcg gaagagcgcc ataatccatg ataaagaaga cagtcataag
     6001 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     6061 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     6121 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6181 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     6241 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     6301 ggtccacg
//
