LOCUS       sequence-208320-        5128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a GST-tagged target protein with 3C protease cleavage 
            site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5128)
  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 5128)
  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-GST-3C-LIC
FEATURES             Location/Qualifiers
     source          1..5128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(182..187,188..203,204..210)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    218..234
                     /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             257..910
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     868..890
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             917..934
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             944..967
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             977..1006
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1031..1048
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1177..1199)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1344..1362)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1430..1534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1535..1603,1604..2395)
                     /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(1753..1772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2566..3154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3055..3074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3308..3325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3398..3475
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3476..4558
                     /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(3495..3514)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4607..4612,4613..4630,4631..4637)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4645..4661
                     /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     4669..4685
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4669..4685
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4681..5128,1..80)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4701..4718)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4701..4717)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4710..4732)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
       61 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
      121 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
      181 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
      241 acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca
      301 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
      361 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
      421 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
      481 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
      541 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
      601 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
      661 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
      721 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
      781 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
      841 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
      901 tcctccaaaa tcggatctgg ttccgcgtgg atcctccgcg gctcttgagg tgctctttca
      961 gggacccggg taccaagaac aaaaactcat ctcagaagag gatctgttgg tacccgggct
     1021 tctcctcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     1081 agctgagttg gctgctgcca ccgctgagca ataactagga attcatcgtg actgactgac
     1141 gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     1201 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     1261 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     1321 gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     1381 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1441 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1501 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1561 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1621 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1681 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1741 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     1801 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1861 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1921 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1981 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2041 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2101 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2161 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2221 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2281 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2341 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2461 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2701 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3181 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3241 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3301 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3361 tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat ggcgcaaaac
     3421 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     3481 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     3541 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     3601 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     3661 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     3721 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     3781 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     3841 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     3901 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     3961 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     4021 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     4081 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     4141 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     4201 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     4261 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     4321 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     4381 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     4441 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     4501 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4561 gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta cactttatgc
     4621 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     4681 atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4741 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4801 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     4861 tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct
     4921 gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc
     4981 tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg
     5041 acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg
     5101 cgaattattt ttgatggcgt tggaatta
//
