LOCUS       sequence-204971-        3612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a His-tagged target protein with 3C protease cleavage 
            site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3612)
  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 3612)
  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-his-3C-LIC-strep
FEATURES             Location/Qualifiers
     source          1..3612
                     /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             126..143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             153..176
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             186..215
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             246..290
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(328..346)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      342..389
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(560..1351)
                     /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(1352..1443)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1491..2229)
                     /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(2439..3521)
                     /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     3483..3502
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3522..3599)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactatag gggaattgtg
       61 agcggataac aattcccctg tagaaataat tttgtttaac tttaataagg agatatacca
      121 tggcacatca ccaccaccat cactccgcgg ctcttgaggt gctctttcag ggacccgggt
      181 accaagaaca aaaactcatc tcagaagagg atctgttggt acccgggctt ctcctcgagt
      241 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
      301 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      361 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
      421 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
      481 aacgaccctg ccctgaaccg acgaccgggt catcgtggcc ggatcttgcg gcccctcggc
      541 ttgaacgaat tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt
      601 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
      661 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
      721 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
      781 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
      841 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
      901 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
      961 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     1021 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     1081 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     1141 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     1201 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
     1261 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     1321 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
     1381 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     1441 aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg gcgctgcgga
     1501 cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg tgaggcaaaa
     1561 cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca gagttcacat
     1621 aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg aatctgacag
     1681 tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt cgctccctct
     1741 tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt tctcccttac
     1801 gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt gtaggtcgtt
     1861 cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg cgccttatcc
     1921 ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact ggcagcagcc
     1981 attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg ctcttgaagt
     2041 gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc tgcgaaagcc
     2101 agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc acctccccag
     2161 gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc tcaagaagat
     2221 cctttgatct tttctactga accgctctag atttcagtgc aatttatctc ttcaaatgta
     2281 gcacctgaag tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg
     2341 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     2401 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2461 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2521 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     2581 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     2641 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     2701 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     2761 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     2821 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     2881 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     2941 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     3001 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     3061 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     3121 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     3181 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     3241 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     3301 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     3361 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     3421 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     3481 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     3541 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     3601 gggatctcga cg
//
