LOCUS       sequence-234028-        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  JOURNAL   Unpublished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            "Unpublished"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"
     protein_bind    8..24
                     /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             47..700
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     658..680
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             707..730
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    755..879
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(904..909,910..927,928..934)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             988..1647
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1054..1073)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1989..2294
                     /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     2173..2192
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2335..2459)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2476..2499
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2569..2591)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2736..2754)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2822..2926
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2927..2995,2996..3787)
                     /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(3145..3164)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3958..4546
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4447..4466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4700..4717
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4790..4867
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4868..5950
                     /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(4887..4906)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5999..6004,6005..6022,6023..6029)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6037..6053
                     /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     6061..6077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6061..6077
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6073..6600
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6093..6110)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6093..6109)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6102..6124)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6702..6707,6708..6723,6724..6730)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 tgtggaattg tgagcggata acaatttcac acaggaaaca gtattcatgt cccctatact
       61 aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga
      121 agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa
      181 gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt
      241 aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg
      301 tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg
      361 tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa
      421 gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg
      481 tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat
      541 ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc
      601 tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg
      661 ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg aagttctgtt
      721 ccaggggccc ctgggatccc cggaattccc atcaacaagt ttgtacaaaa aagctgaacg
      781 agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata aaaaacagac
      841 tacataatac tgtaaaacac aacatatcca gtcactatgg cggccgcatt aggcacccca
      901 ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg
      961 agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt
     1021 gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt
     1081 acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat
     1141 aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg
     1201 gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt
     1261 tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac
     1321 gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg
     1381 gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg
     1441 agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc
     1501 accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt
     1561 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac
     1621 tgcgatgagt ggcagggcgg ggcgtaaaga tctggatccg gcttactaaa agccagataa
     1681 cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc
     1741 cgaagtatgt caaaaagagg tatgctatga agcagcgtat tacagtgaca gttgacagcg
     1801 acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac
     1861 catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg
     1921 gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacagggg
     1981 ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg
     2041 tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca
     2101 gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg
     2161 atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg
     2221 aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt
     2281 tctggggaat ataaatgtca ggctccctta tacacagcca gtctgcaggt cgaccatagt
     2341 gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt
     2401 aatatattga tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtt
     2461 gatgggtcga ctcgagacta caaagacgat gacgacaagt aaactcgagc ggccgcatcg
     2521 tgactgactg acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     2581 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     2641 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     2701 cgatagcgga gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     2761 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     2821 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2881 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2941 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3001 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3061 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3121 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     3181 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3241 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3301 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3361 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3421 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     3481 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3541 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3601 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3661 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3721 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3781 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3841 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3901 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3961 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4021 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4081 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4141 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4201 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4261 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4321 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4381 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4441 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4501 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4561 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4621 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4681 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4741 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga
     4801 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     4861 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     4921 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     4981 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     5041 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     5101 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     5161 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     5221 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     5281 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     5341 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     5401 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     5461 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     5521 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     5581 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     5641 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     5701 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     5761 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     5821 gttgcccgtc tcactggtga aaagaaaaac cacgctagcg cccaatacgc aaaccgcctc
     5881 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     5941 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccccaggctt
     6001 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     6061 caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac
     6121 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     6181 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     6241 ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag
     6301 tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac
     6361 gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc
     6421 acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag
     6481 gaaggccaga cgcgaattat ttttgatggc gttggaatta gcttatcgac tgcacggtgc
     6541 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
     6601 atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc
     6661 cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc
     6721 tcgtataatg
//
