LOCUS       sequence-346299-        6485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SmBit fused to LAF-1 RGG domain with N-terminal MBP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6485)
  AUTHORS   Guan M, Garabedian MV, Leutenegger M, Schuster BS, Good MC, Hammer 
            DA
  TITLE     Incorporation and Assembly of a Light-Emitting Enzymatic Reaction 
            into Model Protein Condensates.
  JOURNAL   Biochemistry. 2021 Oct 14. doi: 10.1021/acs.biochem.1c00373.
  PUBMED    34648259
REFERENCE   2  (bases 1 to 6485)
  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"; journalName: 
            "Biochemistry. 2021 Oct 14. doi: 10.1021/acs.biochem.1c00373."
COMMENT     Sequence Label: MBP-RGG-SmBit-RGG
FEATURES             Location/Qualifiers
     source          1..6485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(77..95)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      91..138
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      175..630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(262..281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     472..493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(745..1560)
                     /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(745..1560)
                     /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     1468..1487
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1552..1571
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1713..2301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2202..2221
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2455..2472
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(2643..3725)
                     /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     3687..3706
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3726..3803)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4009..4028
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4112..4131
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4112..4130
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4131..4155
                     /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             4170..4192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4203..5303
                     /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     5277..5300
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5334..5357
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
       61 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      121 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      181 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      241 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      301 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      361 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      421 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      481 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      541 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      601 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggta cccaggtggc
      661 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      721 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
      781 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
      841 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
      901 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
      961 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     1021 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     1081 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     1141 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     1201 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     1261 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     1321 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     1381 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     1441 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     1501 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     1561 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     1621 cttaacgtga cctaggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     1681 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     1741 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1801 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     1861 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1921 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1981 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2041 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2101 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2161 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2221 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2281 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2341 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2401 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2461 tcagtgagcg aggaagcgga agagcgccat aatccatgat aaagaagaca gtcataagtg
     2521 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     2581 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     2641 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2701 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     2761 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     2821 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     2881 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     2941 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     3001 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     3061 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     3121 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     3181 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     3241 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     3301 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     3361 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     3421 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     3481 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     3541 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     3601 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     3661 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     3721 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     3781 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     3841 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     3901 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     3961 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     4021 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     4081 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     4141 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     4201 tgatgaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg
     4261 gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc
     4321 atccggataa actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca
     4381 ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa
     4441 tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt
     4501 acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca
     4561 aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac
     4621 tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc
     4681 cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta
     4741 aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga
     4801 ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata
     4861 aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca
     4921 aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg
     4981 ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt
     5041 tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc
     5101 tgggtgccgt agcgctgaag tcttacgagg aagagttggt gaaagatccg cgtattgccg
     5161 ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt
     5221 tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg
     5281 aagccctgaa agacgcgcag accggtgggg gagggagtgg cggtggtggc agccttgaag
     5341 tcctctttca ggggccgttg gaatccaatc aatccaataa cggtggctcg ggtaatgccg
     5401 ctctgaatcg tggtggccgc tacgtgccgc cgcatctgcg tggtggcgat ggtggtgcag
     5461 cagcagctgc atctgctggc ggtgatgacc gtcgcggcgg tgcgggcggt ggcggttatc
     5521 gtcgcggcgg tggcaatagt ggtggcggtg gcggtggcgg ttatgatcgt ggttacaatg
     5581 acaaccgtga tgaccgcgat aaccgtggcg gttccggcgg ttacggtcgt gatcgtaatt
     5641 atgaagaccg tggttacaac ggcggtggcg gtggcggtgg caatcgtggc tataacaata
     5701 accgtggtgg cggtggcggt ggctacaatc gccaggatcg tggtgacggt ggcagctcta
     5761 acttttcgcg cggtggctat aataaccgtg atgaaggcag cgacaaccgc ggtagtggcc
     5821 gttcctacaa taacgatcgt cgcgacaatg gtggcgatgg cgaattcggt tctagtggtg
     5881 gtggtggttc tggtggtggt ggttctagtg gtgtgacagg ttatagactt tttgaagaaa
     5941 ttctaggagg aggatcggga ggctctgaat tcgggaagct tatggaatcc aatcaatcca
     6001 ataacggtgg ctcgggtaat gccgctctga atcgtggtgg ccgctacgtg ccgccgcatc
     6061 tgcgtggtgg cgatggtggt gcagcagcag ctgcatctgc tggcggtgat gaccgtcgcg
     6121 gcggtgcggg cggtggcggt tatcgtcgcg gcggtggcaa tagtggtggc ggtggcggtg
     6181 gcggttatga tcgtggttac aatgacaacc gtgatgaccg cgataaccgt ggcggttccg
     6241 gcggttacgg tcgtgatcgt aattatgaag accgtggtta caacggcggt ggcggtggcg
     6301 gtggcaatcg tggctataac aataaccgtg gtggcggtgg cggtggctac aatcgccagg
     6361 atcgtggtga cggtggcagc tctaactttt cgcgcggtgg ctataataac cgtgatgaag
     6421 gcagcgacaa ccgcggtagt ggccgttcct acaataacga tcgtcgcgac aatggtggcg
     6481 atggc
//
