LOCUS       sequence-226702-        6736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows Gateway cloning of gene of interest into bacterial expression
            vector with PreScission Protease cleavable N-terminal GST tag, and a
            retained C-terminal 10xHis tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  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  JOURNAL   Unpublished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 6736)
  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            "Unpublished"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-DEST-His
FEATURES             Location/Qualifiers
     source          1..6736
                     /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"
     protein_bind    966..1090
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1115..1120,1121..1138,1139..1145)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1199..1858
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1265..1284)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2200..2505
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2384..2403
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2546..2670)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2684..2713
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2786..2808)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2953..2971)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3039..3143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3144..3212,3213..4004)
                     /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(3362..3381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4175..4763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4664..4683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4917..4934
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5007..5084
                     /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             5085..6167
                     /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(5104..5123)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6180..6201
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6216..6221,6222..6239,6240..6246)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6254..6270
                     /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     6278..6294
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6278..6294
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6290..6736,1..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(6310..6327)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6310..6326)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6319..6341)
                     /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 ccggaattcc
      961 catcaacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata aatatcaata
     1021 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc
     1081 agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     1141 taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg aagctaaaat
     1201 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     1261 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     1321 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     1381 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga
     1441 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     1501 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     1561 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     1621 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     1681 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
     1741 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     1801 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaaag
     1861 atctggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg
     1921 cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag gtatgctatg
     1981 aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg
     2041 atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg
     2101 ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa
     2161 tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc
     2221 tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg
     2281 cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc
     2341 cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat
     2401 atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa
     2461 aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc aggctccctt
     2521 atacacagcc agtctgcagg tcgaccatag tgactggata tgttgtgttt tacagtatta
     2581 tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat cattttacgt
     2641 ttctcgttca gctttcttgt acaaagtggt tgatgggtcg aggcatcatc atcatcatca
     2701 tcatcatcat cactagtcga ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg
     2761 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2821 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2881 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     2941 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3001 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3061 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3121 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3181 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3241 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3301 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3361 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3421 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3481 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3541 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3601 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3661 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3721 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3781 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3841 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3901 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3961 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4021 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4081 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4141 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4201 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4261 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4321 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4381 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4441 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4501 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4561 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4621 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4681 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4741 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4801 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4861 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4921 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4981 gcggtatttc acaccgcata aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     5041 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     5101 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     5161 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5221 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5281 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5341 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5401 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5461 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5521 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5581 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5641 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5701 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5761 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5821 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5881 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5941 caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     6001 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     6061 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6121 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6181 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6241 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     6301 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6361 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6421 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     6481 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     6541 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     6601 aacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     6661 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     6721 tgatggcgtt ggaatt
//
