LOCUS       sequence-210397-        3579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a StrepII-tagged target protein with 3C protease 
            cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3579)
  AUTHORS   Luna-Vargas MP, Christodoulou E, Alfieri A, van Dijk WJ, Stadnik M, 
            Hibbert RG, Sahtoe DD, Clerici M, Marco VD, Littler D, Celie PH, 
            Sixma TK, Perrakis A
  TITLE     Enabling high-throughput ligation-independent cloning and protein 
            expression for the family of ubiquitin specific proteases.
  JOURNAL   J Struct Biol. 2011 Aug;175(2):113-9. doi: 
            10.1016/j.jsb.2011.03.017. Epub 2011 Mar 29.
  PUBMED    21453775
REFERENCE   2  (bases 1 to 3579)
  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"; doi: 
            "10.1016/j.jsb.2011.03.017"; journalName: "J Struct Biol"; date: 
            "2011-08"; volume: "175"; issue: "2"; pages: "113-9"
COMMENT     Sequence Label: pCDFNKI-strepII-3C-LIC-strep
FEATURES             Location/Qualifiers
     source          1..3579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..52
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        33..51
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    52..76
                     /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             129..152
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             159..182
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             212..256
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(294..312)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      308..355
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(526..1317)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1318..1409)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1457..2196)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(2406..3488)
                     /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     3450..3469
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3489..3566)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactatag gggaattgtg
       61 agcggataac aattcccctg tagaaataat tttgtttaac tttaataagg agatatacca
      121 tggcaagctg gagccacccg cagttcgaaa agggtgcact tgaagtcctc tttcagggac
      181 ccgggtaccc gggcttctcc tcgagtctgg taaagaaacc gctgctgcga aatttgaacg
      241 ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga
      301 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      361 acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
      421 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcatc
      481 gtggccggat cttgcggccc ctcggcttga acgaattgtt agacattatt tgccgactac
      541 cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc
      601 caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata
      661 ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc
      721 ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc
      781 ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg
      841 ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc
      901 tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc
      961 tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg
     1021 ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct
     1081 ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc
     1141 atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc
     1201 atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat
     1261 gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact
     1321 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     1381 atttgaatgt atttagaaaa ataaacaaat agctagctca ctcggtcgct acgctccggg
     1441 cgtgagactg cggcgggcgc tgcggacaca tacaaagtta cccacagatt ccgtggataa
     1501 gcaggggact aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt tttccatagg
     1561 ctccgccctc ctgccagagt tcacataaac agacgctttt ccggtgcatc tgtgggagcc
     1621 gtgaggctca accatgaatc tgacagtacg ggcgaaaccc gacaggactt aaagatcccc
     1681 accgtttccg gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg
     1741 atacctgttc cgcctttctc ccttacggga agtgtggcgc tttctcatag ctcacacact
     1801 ggtatctcgg ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg
     1861 ttcagcccga ctgctgcgcc ttatccggta actgttcact tgagtccaac ccggaaaagc
     1921 acggtaaaac gccactggca gcagccattg gtaactggga gttcgcagag gatttgttta
     1981 gctaaacacg cggttgctct tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga
     2041 tttggttgct gtgctctgcg aaagccagtt accacggtta agtcagttcc ccaactgact
     2101 taaccttcga tcaaaccacc tccccaggtg gttttttcgt ttacagggca aaagattacg
     2161 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctactgaacc gctctagatt
     2221 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     2281 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     2341 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     2401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     2521 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     2581 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     2641 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     2701 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     2761 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     2821 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     2881 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     2941 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     3001 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     3061 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     3121 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     3181 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     3241 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     3301 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     3361 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     3421 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     3481 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     3541 aaggttttgc gccattcgat ggtgtccggg atctcgacg
//
