LOCUS       sequence-233993-        6697 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..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  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  JOURNAL   Unpublished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 6697)
  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            "Unpublished"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
FEATURES             Location/Qualifiers
     source          1..6697
                     /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"
     primer_bind     complement(2536..2558)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2703..2721)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2789..2893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2894..2962,2963..3754)
                     /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(3112..3131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3925..4513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4414..4433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4667..4684
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4757..4834
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4835..5917
                     /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(4854..4873)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5966..5971,5972..5989,5990..5996)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6004..6020
                     /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     6028..6044
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6028..6044
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6040..6567
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6060..6077)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6060..6076)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6069..6091)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(6669..6674,6675..6690,6691..6697)
                     /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 ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg
     2521 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     2581 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     2641 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa
     2701 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     2761 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2821 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2881 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2941 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3001 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3061 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3121 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     3181 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3241 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3301 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3361 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3421 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3481 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3541 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3601 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3661 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3721 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3781 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3841 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3901 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3961 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4021 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4081 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4141 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4201 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4261 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4321 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4381 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4441 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4501 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4561 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4621 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4681 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4741 acaccgcata aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     4801 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     4861 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     4921 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     4981 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5041 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     5101 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     5161 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     5221 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     5281 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     5341 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     5401 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     5461 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     5521 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     5581 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     5641 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     5701 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     5761 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     5821 gctagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     5881 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     5941 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6001 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca
     6061 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     6121 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     6181 ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca
     6241 gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc
     6301 ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc
     6361 attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca
     6421 tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt
     6481 ggaattagct tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     6541 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
     6601 tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
     6661 atgagctgtt gacaattaat catcggctcg tataatg
//
