LOCUS       sequence-121056-        5646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  His-MBP-Smt3-NpuDnaEN102-CBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5646)
  AUTHORS   Guerrero F, Ciragan A, Iwai H
  TITLE     Tandem SUMO fusion vectors for improving soluble protein expression 
            and purification.
  JOURNAL   Protein Expr Purif. 2015 Aug 20. pii: S1046-5928(15)30039-5. doi: 
            10.1016/j.pep.2015.08.019.
  PUBMED    26297996
REFERENCE   2  (bases 1 to 5646)
  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: "Protein 
            Expr Purif. 2015 Aug 20. pii: S1046-5928(15)30039-5. doi: 
            10.1016/j.pep.2015.08.019."
COMMENT     Sequence Label: pLJSRSF7
FEATURES             Location/Qualifiers
     source          1..5646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             99..116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             120..1217
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1191..1214
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1230..1520
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIETHREQIGG"
     CDS             2016..2171
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             2200..2244
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2282..2300)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2296..2343
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2573..3388)
                     /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(2573..3388)
                     /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     3296..3315
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3389..3480)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3496..4245)
                     /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(3506..3523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3746..3765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4441..5523)
                     /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     5485..5504
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5524..5601)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttacttt aataaggaga tataccatgg gcagcagcca tcatcatcat catcacggca
      121 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
      181 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
      241 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
      301 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
      361 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
      421 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
      481 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
      541 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
      601 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
      661 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
      721 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
      781 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
      841 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
      901 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
      961 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     1021 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta
     1081 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     1141 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     1201 tgaaagacgc gcagactaat tcgggtacta gtgactcaga agtcaatcaa gaagctaagc
     1261 cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg tccgatggat
     1321 cttcagaaat cttctttaag atcaaaaaga ccactccttt aagaaggctg atggaagcgt
     1381 tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac ggtattagaa
     1441 ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt attgagactc
     1501 acagagaaca gattggtgga tcccatatgc agtacaaact tatcctgaac ggtaaaaccc
     1561 tgaaaggtga aaccaccacc gaagctgttg acgctgctac cgcggaaaaa gttttcaaac
     1621 agtacgctaa cgacaacggt gttgacggtg aatggaccta cgacgacgct accaaaacct
     1681 acaccgtaac ggaaggatcc tgtttaagct atgaaacgga aatattgaca gtagaatatg
     1741 gattattacc gattggtaaa attgtagaaa agcgcatcga atgtactgtt tatagcgttg
     1801 ataataatgg aaatatttat acacaacctg tagcacaatg gcacgatcgc ggagaacaag
     1861 aggtgtttga gtattgtttg gaagatggtt cattgattcg ggcaacaaaa gaccataagt
     1921 ttatgactgt tgatggtcaa atgttgccaa ttgatgaaat atttgaacgt gaattggatt
     1981 tgatgcgggt tgataatttg ccgaacgaat tcggtaccac aaatcctggt gtatccgctt
     2041 ggcaggtcaa cacagcttat actgcgggac aattggtcac atataacggc aagacgtata
     2101 aatgtttgca gccccacacc tccttggcag gatgggaacc atccaacgtt cctgccttgt
     2161 ggcagcttca ataagcttgc ggccgcactc gagtctggta aagaaaccgc tgctgcgaaa
     2221 tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta ggctgctgcc
     2281 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     2341 ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa
     2401 ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga accgacgaca
     2461 agctgacgac cgggtctccg caagtggcac ttttcgggga aatgtgcgcg gaacccctat
     2521 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2581 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2641 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2701 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2761 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2821 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2881 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2941 cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     3001 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     3061 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     3121 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     3181 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     3241 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     3301 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     3361 aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat tgaagcattt
     3421 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3481 taggcatgca gcgctcttcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac
     3541 tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg gttatccaca gaatcagggg
     3601 ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc aacgccgcag
     3661 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagccag
     3721 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3781 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3841 ggaagcgtgg cgctttctca tagctcacgc tgttggtatc tcagttcggt gtaggtcgtt
     3901 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3961 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4021 attggtaact gatttagagg actttgtctt gaagttatgc acctgttaag gctaaactga
     4081 aagaacagat tttggtgagt gcggtcctcc aacccactta ccttggttca aagagttggt
     4141 agctcagcga accttgagaa aaccaccgtt ggtagcggtg gtttttcttt atttatgaga
     4201 tgatgaatca atcggtctat caagtcaacg aacagctatt ccgttactct agatttcagt
     4261 gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt
     4321 ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4381 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4441 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4501 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4561 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4621 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4681 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     4741 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4801 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4861 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4921 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4981 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5041 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5101 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5161 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5221 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5281 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5341 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5401 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5461 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5521 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5581 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5641 ggaaat
//
