LOCUS       sequence-226705-        6730 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 FLAG tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  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  JOURNAL   Unpublished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 6730)
  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            "Unpublished"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-FLAG
FEATURES             Location/Qualifiers
     source          1..6730
                     /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             2687..2710
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2780..2802)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2947..2965)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3033..3137
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3138..3206,3207..3998)
                     /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(3356..3375)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4169..4757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4658..4677
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4911..4928
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5001..5078
                     /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             5079..6161
                     /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(5098..5117)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6174..6195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6210..6215,6216..6233,6234..6240)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6248..6264
                     /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     6272..6288
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6272..6288
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6284..6730,1..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(6304..6321)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6304..6320)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6313..6335)
                     /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 actcgagact acaaagacga
     2701 tgacgacaag taaactcgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt
     2761 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2821 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2881 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     2941 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3001 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3061 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3121 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3181 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3241 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3301 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3361 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     3421 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3481 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3541 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3601 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3661 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3721 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3781 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3841 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3901 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3961 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4021 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4081 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4141 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4201 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4261 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4321 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4381 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4441 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4501 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4561 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4621 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4681 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4741 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4801 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4861 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4921 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4981 tttcacaccg cataaattcc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat
     5041 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     5101 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     5161 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     5221 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     5281 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     5341 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     5401 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     5461 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     5521 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     5581 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     5641 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     5701 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     5761 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     5821 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     5881 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgtcaacca
     5941 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     6001 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     6061 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     6121 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     6181 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     6241 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga
     6301 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     6361 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     6421 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     6481 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     6541 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta
     6601 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     6661 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     6721 cgttggaatt
//
