LOCUS       sequence-243311-        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 Strep-II 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  JOURNAL   Unpublished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            "Unpublished"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-Strep2
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    784..908
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(933..938,939..956,957..963)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1017..1676
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1083..1102)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2018..2323
                     /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     2202..2221
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2364..2488)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2505..2528
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2598..2620)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2765..2783)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2851..2955
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2956..3024,3025..3816)
                     /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(3174..3193)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3987..4575
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4476..4495
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4729..4746
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4819..4896
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4897..5979
                     /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(4916..4935)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6028..6033,6034..6051,6052..6058)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6066..6082
                     /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     6090..6106
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6090..6106
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6102..6629
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6122..6139)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6122..6138)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6131..6153)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatcccc ggaattccca
      781 tcaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata
      841 ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
      901 tcactatggc ggccgcatta ggcaccccag gctttacact ttatgcttcc ggctcgtata
      961 atgtgtggat tttgagttag gatccgtcga gattttcagg agctaaggaa gctaaaatgg
     1021 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
     1081 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
     1141 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
     1201 ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc
     1261 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
     1321 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc
     1381 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
     1441 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
     1501 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
     1561 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
     1621 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaaagat
     1681 ctggatccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg
     1741 gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa
     1801 gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat
     1861 gtcaatatct ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc
     1921 gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg
     1981 aacggctctt ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta
     2041 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     2101 cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg
     2161 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     2221 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     2281 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     2341 acacagccag tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg
     2401 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     2461 ctcgttcagc tttcttgtac aaagtggttg atgggtcgac tcgatggagc cacccgcagt
     2521 tcgaaaagta aactcgagcg gccgcatcgt gactgactga cgatctgcct cgcgcgtttc
     2581 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     2641 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     2701 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga
     2761 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2821 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2881 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2941 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3001 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3061 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3121 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3181 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     3241 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3301 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3361 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3421 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3481 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3541 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3601 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3661 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3721 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3781 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3841 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3901 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3961 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4021 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4081 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4141 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4201 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4261 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4321 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4381 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4441 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4501 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4561 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4621 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4681 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4741 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4801 tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     4861 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     4921 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     4981 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     5041 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     5101 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     5161 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     5221 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     5281 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     5341 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     5401 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     5461 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     5521 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     5581 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     5641 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     5701 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     5761 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     5821 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     5881 acgctagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     5941 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     6001 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6061 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt
     6121 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     6181 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     6241 gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac
     6301 cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg
     6361 tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac gtgacctatc
     6421 ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca
     6481 catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg
     6541 ttggaattag cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg
     6601 aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc
     6661 actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg
     6721 aaatgagctg
//
