LOCUS       sequence-177271-        3903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For constructing SUMO-fusion proteins with N-terminal StrepII tag 
            and C-terminal His tag using ""blunt-end"" cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3903)
  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 3903)
  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: pTS2
FEATURES             Location/Qualifiers
     source          1..3903
                     /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..123
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             424..441
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             457..501
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(539..557)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      553..600
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(830..1645)
                     /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(830..1645)
                     /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     1553..1572
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1646..1737)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1753..2502)
                     /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(1763..1780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2003..2022)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2698..3780)
                     /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     3742..3761
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3781..3858)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg agccacgtgt ggtcgcaccc tcaattcgag
      121 aaaggatcct gggaagttaa accggaaacc cacatcaacc tgaaagtttc tgacggttct
      181 tctgaaatct tcttcaaaat caaaaaaacc accccgctgc gtcgtctgat ggaagctttc
      241 gctaaacgtc agggtaaaga aatggactct ctgcgtttcc tgtacgacgg tatccgtatc
      301 caggctgacc agaccccgga agacctggac atggaagaca acgacatcat cgaagctcac
      361 cgtgaacagc tgggtggtgc taccatggaa ttcgggcccg atatcggtac cctcgaggat
      421 atccaccatc atcaccacca ttgagtcgag tctggtaaag aaaccgctgc tgcgaaattt
      481 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
      541 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      601 ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg
      661 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgacaagc
      721 tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      781 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
      841 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
      901 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
      961 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1021 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1081 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1141 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1201 gacgaaatac gcggtcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     1261 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     1321 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     1381 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     1441 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     1501 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     1561 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     1621 acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc
     1681 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1741 gcatgcagcg ctcttccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     1801 ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt atccacagaa tcaggggata
     1861 aagccggaaa gaacatgtga gcaaaaagca aagcaccgga agaagccaac gccgcaggcg
     1921 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagccagagg
     1981 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2041 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2101 agcgtggcgc tttctcatag ctcacgctgt tggtatctca gttcggtgta ggtcgttcgc
     2161 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2221 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccatt
     2281 ggtaactgat ttagaggact ttgtcttgaa gttatgcacc tgttaaggct aaactgaaag
     2341 aacagatttt ggtgagtgcg gtcctccaac ccacttacct tggttcaaag agttggtagc
     2401 tcagcgaacc ttgagaaaac caccgttggt agcggtggtt tttctttatt tatgagatga
     2461 tgaatcaatc ggtctatcaa gtcaacgaac agctattccg ttactctaga tttcagtgca
     2521 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc
     2581 atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     2641 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     2701 ctgcccgctt tccagtcggg aaacctgtcg tgccagttgc attaatgaat cggccaacgc
     2761 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     2821 gggcaacagt tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     2881 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     2941 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     3001 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     3061 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     3121 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     3181 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     3241 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     3301 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     3361 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     3421 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     3481 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     3541 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     3601 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     3661 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     3721 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     3781 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     3841 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     3901 aat
//
