LOCUS       sequence-373100-        6076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express in E. coli human gelsolin segments 4 through 6 with 
            N-terminal 6xHis and MBP tags..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6076)
  AUTHORS   Ceron RH, Carman PJ, Rebowski G, Boczkowska M, Heuckeroth RO, 
            Dominguez R
  TITLE     A solution to the long-standing problem of actin expression and 
            purification.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Oct 11;119(41):e2209150119. doi: 
            10.1073/pnas.2209150119. Epub 2022 Oct 5.
  PUBMED    36197995
REFERENCE   2  (bases 1 to 6076)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-10-11"; volume: "119(41):e2209150119. 
            doi"; pages: " 10.1073/pnas.2209150119. Epub 2022 Oct 5"
COMMENT     Sequence Label: His-MBP_G4G6 in pRSF
FEATURES             Location/Qualifiers
     source          1..6076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(52..143)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(159..908)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(169..186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(409..428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1104..2186)
                     /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     2148..2167
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2187..2264)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        2310..2328
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2329..2353
                     /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)."
     CDS             2409..2426
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2463..3563
                     /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
                     WYAVRTAVINAASGRQTVDAALAAAQT"
     CDS             3612..3623
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     regulatory      3809..3818
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        4787..4805
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4806..4830
                     /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)."
     CDS             4939..4983
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5021..5039)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5035..5082
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(join(5312..6076,1..51))
                     /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(join(5312..6076,1..51))
                     /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     6035..6054
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct
       61 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
      121 atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga
      181 ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat
      241 acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc
      301 accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc
      361 acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg
      421 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
      481 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt
      541 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      601 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      661 acttatcgcc actggcagca gccattggta actgatttag aggactttgt cttgaagtta
      721 tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac
      781 ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg
      841 gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct
      901 attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
      961 cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc
     1021 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     1081 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1141 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     1201 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     1261 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     1321 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     1381 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     1441 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     1501 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     1561 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     1621 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     1681 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     1741 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     1801 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     1861 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     1921 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     1981 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     2041 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     2101 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     2161 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     2221 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     2281 tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg aattgtgagc
     2341 ggataacaat tcccctgtag aaataatttt gtttaacttt aataaggaga tataccatgg
     2401 gcagcagcca tcaccatcat caccacagcc aggatccgaa ttcgagctcg gcgcgcctgc
     2461 agatgaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg
     2521 gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc
     2581 atccggataa actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca
     2641 ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa
     2701 tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt
     2761 acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca
     2821 aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac
     2881 tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc
     2941 cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta
     3001 aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga
     3061 ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata
     3121 aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca
     3181 aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg
     3241 ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt
     3301 tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc
     3361 tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg
     3421 ccactatgga aaacgcccag aaaggtgaaa tcatgcctaa catcccgcag atgtccgctt
     3481 tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg
     3541 cagccctggc agccgcgcag actaatgcgg ccgcgaacaa caacaacaat aacaataaca
     3601 acaacctcgg gatcgaggga aggatttcag aattcggatc ccatatggag ctcggtaccc
     3661 tcgaggccgc ccagcacggc atggatgacg atggcacagg ccagaaacag atctggagaa
     3721 tcgaaggttc caacaaggtg cccgtggacc ctgccacata tggacagttc tatggaggcg
     3781 acagctacat cattctgtac aactaccgcc atggtggccg ccaggggcag ataatctata
     3841 actggcaggg tgcccagtct acccaggatg aggtcgctgc atctgccatc ctgactgctc
     3901 agctggatga ggagctggga ggtacccctg tccagagccg tgtggtccaa ggcaaggagc
     3961 ccgcccacct catgagcctg tttggtggga agcccatgat catctacaag ggcggcacct
     4021 cccgcgaggg cgggcagaca gcccctgcca gcacccgcct cttccaggtc cgcgccaaca
     4081 gcgctggagc cacccgggct gttgaggtat tgcctaaggc tggtgcactg aactccaacg
     4141 atgcctttgt tctgaaaacc ccctcagccg cctacctgtg ggtgggtaca ggagccagcg
     4201 aggcagagaa gacgggggcc caggagctgc tcagggtgct gcgggcccaa cctgtgcagg
     4261 tggcagaagg cagcgagcca gatggcttct gggaggccct gggcgggaag gctgcctacc
     4321 gcacatcccc acggctgaag gacaagaaga tggatgccca tcctcctcgc ctctttgcct
     4381 gctccaacaa gattggacgt tttgtgatcg aagaggttcc tggtgagctc atgcaggaag
     4441 acctggcaac ggatgacgtc atgcttctgg acacctggga ccaggtcttt gtctgggttg
     4501 gaaaggattc tcaagaagaa gaaaagacag aagccttgac ttctgctaag cggtacatcg
     4561 agacggaccc agccaatcgg gatcggcgga cgcccatcac cgtggtgaag caaggctttg
     4621 agcctccctc ctttgtgggc tggttccttg gctgggatga tgattactgg tctgtggacc
     4681 ccttggacag ggccatggct gagctggctg cctgaaagct tgcggccgca taatgcttaa
     4741 gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat acgactcact
     4801 ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta agtataagaa
     4861 ggagatatac atatggcaga tctcaattgg atatcggccg gccacgcgat cgctgacgtc
     4921 ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac
     4981 tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca
     5041 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt
     5101 gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca
     5161 taagcggcta tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc
     5221 aagtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5281 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     5341 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     5401 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     5461 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     5521 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     5581 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     5641 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc
     5701 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     5761 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     5821 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     5881 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     5941 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     6001 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     6061 aatcagcatc catgtt
//
