LOCUS       sequence-237583-        6882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Solubly expresses monomeric streptavidin containing S25H mutation in
            E. coli as an MBP fusion. App Microbiol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6882)
  AUTHORS   Demonte D, Dundas CM, Park S
  TITLE     Expression and purification of soluble monomeric streptavidin in 
            Escherichia coli.
  JOURNAL   Appl Microbiol Biotechnol. 2014;98(14):6285-95. doi: 
            10.1007/s00253-014-5682-y. Epub 2014 Apr 2.
  PUBMED    24691867
REFERENCE   2  (bases 1 to 6882)
  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: "Appl 
            Microbiol Biotechnol."; date: "2014"; volume: "98(14):6285-95. doi";
            pages: " 10.1007/s00253-014-5682-y. Epub 2014 Apr 2"
COMMENT     Sequence Label: pET-MBP-mSA2
FEATURES             Location/Qualifiers
     source          1..6882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     130..149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        130..148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    149..173
                     /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             188..210
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             229..246
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             256..273
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             277..1377
                     /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
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1234..1243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1351..1374
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1426..1437
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1459..1479
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1480..1821
                     /codon_start=1
                     /product="monomeric streptavidin/rhizavidin hybrid (Lim et 
                     al., 2013)"
                     /label=mSA
                     /translation="AEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPY
                     TLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATE
                     QGQDTFTKVK"
     CDS             1843..1866
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1876..1893
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1946..1964)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1960..2007
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2044..2499
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2131..2150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2341..2362
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2591..3406)
                     /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(2591..3406)
                     /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     3314..3333
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3398..3417
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3528..4116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4017..4036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4270..4287
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4302..4441
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4367..4386)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4527..4549
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4543..4734)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5543..6625)
                     /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     6587..6606
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6626..6703)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
       61 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      121 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      181 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
      241 catcacagca gcggcctggt gccgcgcggc agccatatga aaatcgaaga aggtaaactg
      301 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
      361 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
      421 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
      481 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
      541 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
      601 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
      661 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
      721 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
      781 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
      841 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
      901 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
      961 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
     1021 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
     1081 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
     1141 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     1201 gaggaagagt tggcgaaaga tccacgtatt gccgccacca tggaaaacgc ccagaaaggt
     1261 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     1321 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat
     1381 tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga gggaagggga
     1441 ggatccgaat tcgctagcga gaacctgtat ttccagggcg cggaagcggg tatcaccggc
     1501 acgtggtaca accagcatgg ttctaccttc accgttaccg cgggtgcgga cggtaacctg
     1561 accggtcagt acgaaaaccg tgcgcagggc actggttgcc agaactctcc gtacaccctg
     1621 accggtcgtt acaacggtac caaactggaa tggcgtgttg aatggaacaa ctctaccgaa
     1681 aactgccact ctcgtaccga atggcgtggt cagtaccagg gtggtgcgga agcgcgtatc
     1741 aacacccagt ggaacctgac ctacgaaggt ggttctggtc cggcgaccga acagggtcag
     1801 gacaccttca ccaaagttaa accgtctgcg gcgtctggat ccgactacaa ggacgatgac
     1861 gacaagtaac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1921 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1981 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2041 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2101 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2161 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2221 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2281 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2341 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2401 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2461 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2521 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2581 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2641 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2701 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     2761 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2821 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2881 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2941 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3001 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3061 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3121 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3181 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3241 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3301 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3361 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3421 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3481 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3541 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3601 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3661 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3721 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3781 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3841 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3901 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3961 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4021 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4081 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4141 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4201 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4261 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4321 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4381 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4441 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4501 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4561 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4621 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4681 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4741 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4801 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4861 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4921 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4981 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5041 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5101 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5161 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5221 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5281 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5341 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5401 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5461 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5521 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5581 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5641 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5701 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5761 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5821 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5881 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5941 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6001 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6061 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6121 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6181 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6241 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6301 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6361 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6421 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6481 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6541 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6601 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6661 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6721 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6781 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6841 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cg
//
