LOCUS       sequence-177269-        5016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For constructing MBP-SUMO-fusion proteins with N-terminal His tag 
            and C-terminal strep II tag using ""blunt-end"" cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5016)
  AUTHORS   Zhang Y, Fan Y
  TITLE     A Mutant Sumo Facilitates Quick Plasmid Construction for Expressing 
            Proteins with Native N-termini After Tag Removal.
  JOURNAL   Mol Biotechnol. 2017 May;59(4-5):159-167. doi: 
            10.1007/s12033-017-9998-6.
  PUBMED    28349302
REFERENCE   2  (bases 1 to 5016)
  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: "Mol 
            Biotechnol."; date: "2017-05"; volume: "59(4-5):159-167. doi"; 
            pages: " 10.1007/s12033-017-9998-6"
COMMENT     Sequence Label: pTMS1
FEATURES             Location/Qualifiers
     source          1..5016
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..1224
                     /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
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPAAAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYAAGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDAALAAAQT"
     CDS             1531..1554
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1570..1614
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1652..1670)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1666..1713
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1943..2758)
                     /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(1943..2758)
                     /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     2666..2685
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2759..2850)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2866..3615)
                     /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(2876..2893)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3116..3135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3811..4893)
                     /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     4855..4874
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4894..4971)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg agccacgtgc atcaccatca tcaccacgga
      121 tcctttaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
      181 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
      241 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
      301 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
      361 atcaccccgg ccgcagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
      421 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
      481 aaagacctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
      541 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
      601 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg cagccggcaa gtacgacatt
      661 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
      721 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
      781 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
      841 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
      901 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag
      961 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1021 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     1081 gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     1141 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     1201 gcagccctgg cagccgcgca gacttacgta agatcctggg aagttaaacc ggaaacccac
     1261 atcaacctga aagtttctga cggttcttct gaaatcttct tcaaaatcaa aaaaaccacc
     1321 ccgctgcgtc gtctgatgga agctttcgct aaacgtcagg gtaaagaaat ggactctctg
     1381 cgtttcctgt acgacggtat ccgtatccag gctgaccaga ccccggaaga cctggacatg
     1441 gaagacaacg acatcatcga agctcaccgt gaacagctgg gtggtgctac catggaattc
     1501 gggcccgata tcggtaccct cgaggatatc tggtcgcacc ctcaattcga gaaatgagtc
     1561 gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact
     1621 agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt
     1681 ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac
     1741 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
     1801 atttaacgac cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac
     1861 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1921 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     1981 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2041 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2101 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2161 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2221 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2281 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag
     2341 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2401 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2461 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2521 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2581 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2641 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2701 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac
     2761 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2821 tatttgaatg tatttagaaa aataaacaaa taggcatgca gcgctcttcc gcttcctcgc
     2881 tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag
     2941 cggtaatacg gttatccaca gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa
     3001 gcaaagcacc ggaagaagcc aacgccgcag gcgtttttcc ataggctccg cccccctgac
     3061 gagcatcaca aaaatcgacg ctcaagccag aggtggcgaa acccgacagg actataaaga
     3121 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3181 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3241 tgttggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3301 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3361 agacacgact tatcgccact ggcagcagcc attggtaact gatttagagg actttgtctt
     3421 gaagttatgc acctgttaag gctaaactga aagaacagat tttggtgagt gcggtcctcc
     3481 aacccactta ccttggttca aagagttggt agctcagcga accttgagaa aaccaccgtt
     3541 ggtagcggtg gtttttcttt atttatgaga tgatgaatca atcggtctat caagtcaacg
     3601 aacagctatt ccgttactct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     3661 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     3721 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3781 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3841 tcgtgccagt tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3901 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agttgattgc ccttcaccgc
     3961 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4021 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4081 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4141 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4201 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4261 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4321 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4381 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4441 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4501 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4561 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4621 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4681 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4741 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4801 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4861 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4921 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4981 gacgctctcc cttatgcgac tcctgcatta ggaaat
//
