LOCUS       sequence-226706-        5020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows cloning of gene of interest into bacterial expression vector 
            with PreScission Protease cleavable N-terminal GST tag, and a 
            retained N-terminal 10x His tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5020)
  TITLE     Bacterial expression plasmids for production of recombinant proteins
            with a PreScission Protease cleavable GST tag and additional small 
            affinity tags
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5020)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pGEX6P1-N-His
FEATURES             Location/Qualifiers
     source          1..5020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             951..980
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1070..1092)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1237..1255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1323..1427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1428..1496,1497..2288)
                     /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(1646..1665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2459..3047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2948..2967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3201..3218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3291..3368
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3369..4451
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3388..3407)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4464..4485
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4500..4505,4506..4523,4524..4530)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4538..4554
                     /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     4562..4578
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4562..4578
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4574..5020,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4594..4611)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4594..4610)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4603..4625)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc catcatcatc
      961 atcatcatca tcatcatcac cgatccccgg aattcccggg tcgactcgag cggccgcatc
     1021 gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1081 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1141 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     1201 gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     1261 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     1321 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1381 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1441 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1501 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1561 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1621 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     1681 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1741 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1801 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1861 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1921 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     1981 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2041 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2101 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2161 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2221 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2281 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2341 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2401 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2461 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2521 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2581 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2641 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2701 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2761 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2821 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2881 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2941 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3001 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3061 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg
     3301 aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     3361 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     3421 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     3481 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     3541 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     3601 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     3661 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     3721 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     3781 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     3841 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     3901 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     3961 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     4021 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     4081 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     4141 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     4201 acagctcatg ttatatcccg ccgtcaacca ccatcaaaca ggattttcgc ctgctggggc
     4261 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     4321 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     4381 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4441 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     4501 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     4561 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
     4621 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4681 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4741 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
     4801 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
     4861 cgatgcgccc atctacacca acgtaaccta tcccattacg gtcaatccgc cgtttgttcc
     4921 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
     4981 ggaaggccag acgcgaatta tttttgatgg cgttggaatt
//
