LOCUS       sequence-346289-        6902 bp    DNA     circular SYN 15-APR-2025
DEFINITION  LgBit 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 6902)
  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 6902)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-LgBit-RGG
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             729..746
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     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"
     CDS             complement(1995..2810)
                     /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(1995..2810)
                     /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     2718..2737
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2802..2821
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2963..3551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3452..3471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3705..3722
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(3893..4975)
                     /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     4937..4956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4976..5053)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5259..5278
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5362..5381
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5362..5380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5381..5405
                     /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             5420..5442
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5453..5470
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      5467..5476
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5483..6583
                     /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     6557..6580
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             6614..6637
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 gttacggtcg tgatcgtaat tatgaagacc gtggttacaa cggcggtggc ggtggcggtg
       61 gcaatcgtgg ctataacaat aaccgtggtg gcggtggcgg tggctacaat cgccaggatc
      121 gtggtgacgg tggcagctct aacttttcgc gcggtggcta taataaccgt gatgaaggca
      181 gcgacaaccg cggtagtggc cgttcctaca ataacgatcg tcgcgacaat ggtggcgatg
      241 gcgaattcgt ttttacgctt gaagatttcg ttggtgattg ggagcaaact gcggcatata
      301 atttggacca agttttggaa caaggtggtg taagtagtct tcttcagaac ttagctgtct
      361 ccgtgacacc cattcagcgc atcgttagat cgggtgaaaa cgccctgaag atagatattc
      421 acgttattat tccctatgag ggcttaagtg cagatcaaat ggcccaaatc gaggaggttt
      481 tcaaggtcgt gtatccagtg gacgatcacc atttcaaagt cattttaccg tatgggactt
      541 tagtaattga cggagtaact cccaatatgt tgaactattt tggaagacca tacgagggca
      601 tagcggtttt cgatggtaaa aagattacgg tgacgggtac gctttggaat ggtaacaaaa
      661 ttatagacga gcggttaata accccggatg ggtcaatgtt attcagagtc actattaaca
      721 gtgaattctt agtacctcgc gggtcaatgg aatccaatca atccaataac ggtggctcgg
      781 gtaatgccgc tctgaatcgt ggtggccgct acgtgccgcc gcatctgcgt ggtggcgatg
      841 gtggtgcagc agcagctgca tctgctggcg gtgatgaccg tcgcggcggt gcgggcggtg
      901 gcggttatcg tcgcggcggt ggcaatagtg gtggcggtgg cggtggcggt tatgatcgtg
      961 gttacaatga caaccgtgat gaccgcgata accgtggcgg ttccggcggt tacggtcgtg
     1021 atcgtaatta tgaagaccgt ggttacaacg gcggtggcgg tggcggtggc aatcgtggct
     1081 ataacaataa ccgtggtggc ggtggcggtg gctacaatcg ccaggatcgt ggtgacggtg
     1141 gcagctctaa cttttcgcgc ggtggctata ataaccgtga tgaaggcagc gacaaccgcg
     1201 gtagtggccg ttcctacaat aacgatcgtc gcgacaatgg tggcgatggc ctcgagcacc
     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 tttcggggta cccaggtggc acttttcggg
     1921 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1981 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2041 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     2101 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     2161 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     2221 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     2281 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     2341 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     2401 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2461 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     2521 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2581 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2641 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     2701 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     2761 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     2821 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     2881 cctaggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2941 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3001 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3061 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     3121 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3181 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3241 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3301 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3361 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3421 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3481 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3541 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3601 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3661 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3721 aggaagcgga agagcgccat aatccatgat aaagaagaca gtcataagtg cggcgacgat
     3781 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     3841 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     3901 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3961 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4021 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4081 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4141 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4201 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4261 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4321 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4381 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4441 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4501 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4561 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4621 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4681 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     4741 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     4801 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     4861 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     4921 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     4981 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5041 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5101 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5161 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5221 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     5281 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     5341 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     5401 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgcaccatca
     5461 ccatcaccat ggcggcggat ctatgaaaat cgaagaaggt aaactggtaa tctggattaa
     5521 cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg
     5581 aattaaagtc accgttgagc atccggataa actggaagag aaattcccac aggttgcggc
     5641 aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca
     5701 atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt
     5761 tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc
     5821 gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat
     5881 cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca
     5941 agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga
     6001 aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct
     6061 gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat
     6121 cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg
     6181 gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg
     6241 tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa
     6301 caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc
     6361 ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggt
     6421 gaaagatccg cgtattgccg ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa
     6481 catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag
     6541 cggtcgtcag actgtcgatg aagccctgaa agacgcgcag accggtgggg gagggagtgg
     6601 cggtggtggc agccttgaag tcctctttca ggggccgggt atggaatcca atcaatccaa
     6661 taacggtggc tcgggtaatg ccgctctgaa tcgtggtggc cgctacgtgc cgccgcatct
     6721 gcgtggtggc gatggtggtg cagcagcagc tgcatctgct ggcggtgatg accgtcgcgg
     6781 cggtgcgggc ggtggcggtt atcgtcgcgg cggtggcaat agtggtggcg gtggcggtgg
     6841 cggttatgat cgtggttaca atgacaaccg tgatgaccgc gataaccgtg gcggttccgg
     6901 cg
//
