LOCUS       sequence-208319-        5152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a GST-6xHis-tagged target protein with 3C protease 
            cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5152)
  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 5152)
  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: pGEXNKI-his-GST-3C-LIC
FEATURES             Location/Qualifiers
     source          1..5152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(178..183,184..199,200..206)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    214..230
                     /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             253..906
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     864..886
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             913..930
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             937..954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             964..987
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             997..1026
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1051..1068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1197..1219)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1364..1382)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1450..1554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1555..1623,1624..2415)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1773..1792)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2586..3174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3075..3094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3328..3345
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3418..3495
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3496..4578
                     /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     complement(3515..3534)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4627..4632,4633..4650,4651..4657)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4665..4681
                     /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)."
     primer_bind     4689..4705
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4689..4705
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4701..5152,1..76)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4721..4738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4721..4737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4730..4752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
       61 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
      121 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg
      181 acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag
      241 gaaacagtat tcatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
      301 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
      361 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
      421 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
      481 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
      541 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
      601 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
      661 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
      721 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
      781 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
      841 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
      901 ccaaaatcgg atctggttcc gcgtggatcc atggcacatc accaccacca tcactccgcg
      961 gctcttgagg tgctctttca gggacccggg taccaagaac aaaaactcat ctcagaagag
     1021 gatctgttgg tacccgggct tctcctcgag caccaccacc accaccactg agatccggct
     1081 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagga
     1141 attcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     1201 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     1261 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     1321 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta
     1381 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1441 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1501 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1561 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1621 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1681 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1741 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     1801 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1861 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1921 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1981 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2041 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2101 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2161 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2221 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2281 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2341 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2401 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2461 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2521 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2581 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2641 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2701 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2761 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2821 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2881 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2941 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3001 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3061 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3121 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3181 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3241 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3301 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3361 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac
     3421 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     3481 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     3541 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     3601 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     3661 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     3721 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     3781 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     3841 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     3901 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     3961 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     4021 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     4081 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     4141 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     4201 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     4261 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     4321 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     4381 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     4441 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     4501 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4561 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     4621 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4681 atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac
     4741 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4801 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4861 gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct
     4921 ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc
     4981 acggttacga tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt
     5041 ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct
     5101 ggctacagga aggccagacg cgaattattt ttgatggcgt tggaattagc tt
//
