LOCUS       sequence-210396-        3613 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 3613)
  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 3613)
  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..3613
                     /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..2230)
                     /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(2440..3522)
                     /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     3484..3503
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3523..3600)
                     /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 gttaagtcag ttccccaact gacttaacct tcgatcaaac cacctcccca
     2161 ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga
     2221 tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct cttcaaatgt
     2281 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
     2341 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     2401 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2521 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     2581 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     2641 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     2701 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     2761 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     2821 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     2881 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     2941 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     3001 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     3061 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     3121 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     3181 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3241 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3301 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3361 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3421 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     3481 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     3541 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     3601 cgggatctcg acg
//
