LOCUS       pETNKI-strepII-3C-LIC-kan               5309 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of a StrepII-tagged target protein with 3C protease 
            cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    pETNKI-strepII-3C-LIC-kan
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5309)
  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 5309)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5309
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      7..462
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(554..1369)
                     /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     1277..1296
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1361..1380
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1491..2079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1980..1999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2233..2250
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2265..2404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2330..2349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2490..2512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2506..2697)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3506..4588)
                     /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     4550..4569
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4589..4666)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4872..4891
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4975..4994
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4975..4993
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4994..5018
                     /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)."
     RBS             5033..5055
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5071..5094
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5101..5124
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5148..5165
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5218..5236)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5232..5279
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      421 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
      481 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      541 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
      601 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
      661 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
      721 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
      781 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
      841 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
      901 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
      961 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     1021 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     1081 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     1141 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     1201 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     1261 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     1321 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     1381 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     1441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1621 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     1681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2101 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2161 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2221 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     2281 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     2341 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     2401 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2461 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2521 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     2581 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     2641 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     2701 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     2761 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     2821 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     2881 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     2941 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     3001 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     3061 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     3121 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     3181 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     3241 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     3301 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     3361 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     3421 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     3481 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3541 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     3601 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     3661 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     3721 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     3781 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     3841 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     3901 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     3961 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4021 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     4081 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     4141 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     4201 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     4261 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     4321 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     4381 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     4441 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     4501 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     4561 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     4621 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     4681 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     4741 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     4801 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     4861 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     4921 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     4981 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     5041 actttaagaa ggagatatac catggcaagc tggagccacc cgcagttcga aaagggtgca
     5101 cttgaagtcc tctttcaggg acccgggtac ccgggcttct cctcgagcac caccaccacc
     5161 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     5221 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     5281 tgaaaggagg aactatatcc ggattggcg
//