LOCUS       sequence-234027-        6735 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 HA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6735)
  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  JOURNAL   Unpublished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 6735)
  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            "Unpublished"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-HA
FEATURES             Location/Qualifiers
     source          1..6735
                     /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             2479..2505
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2574..2596)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2741..2759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2827..2931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2932..3000,3001..3792)
                     /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(3150..3169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3963..4551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4452..4471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4705..4722
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4795..4872
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4873..5955
                     /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(4892..4911)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6004..6009,6010..6027,6028..6034)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6042..6058
                     /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     6066..6082
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6066..6082
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6078..6605
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6098..6115)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6098..6114)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6107..6129)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6707..6712,6713..6728,6729..6735)
                     /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 ctcgaagcta cccatacgat gttccagatt acgcttaact cgagcggccg
     2521 catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     2581 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     2641 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     2701 cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt
     2761 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     2821 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2881 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2941 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3001 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3061 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3121 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     3181 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3241 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3301 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3361 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3421 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     3481 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3541 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3601 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3661 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3721 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3781 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3841 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3901 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3961 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4021 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4081 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4141 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4201 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4261 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4321 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4381 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4441 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4501 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4561 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4621 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4681 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4741 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc
     4801 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     4861 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     4921 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     4981 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     5041 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     5101 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     5161 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     5221 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     5281 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     5341 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     5401 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     5461 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     5521 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     5581 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     5641 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     5701 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     5761 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     5821 cagctgttgc ccgtctcact ggtgaaaaga aaaaccacgc tagcgcccaa tacgcaaacc
     5881 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5941 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca
     6001 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6061 tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc
     6121 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     6181 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     6241 tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc
     6301 tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg
     6361 gttacgatgc gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg
     6421 ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc
     6481 tacaggaagg ccagacgcga attatttttg atggcgttgg aattagctta tcgactgcac
     6541 ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt
     6601 cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt
     6661 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca
     6721 tcggctcgta taatg
//
