LOCUS       sequence-234026-        6736 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 10xHis tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  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  JOURNAL   Unpublished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 6736)
  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            "Unpublished"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-His
FEATURES             Location/Qualifiers
     source          1..6736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(111..116,117..134,135..141)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             195..854
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(261..280)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1196..1501
                     /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     1380..1399
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1542..1666)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1680..1709
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1782..1804)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1949..1967)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2035..2139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2140..2208,2209..3000)
                     /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(2358..2377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3171..3759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3660..3679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3913..3930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4003..4080
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4081..5163
                     /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(4100..4119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5212..5217,5218..5235,5236..5242)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5250..5266
                     /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     5274..5290
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5274..5290
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5286..5813
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5306..5323)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5306..5322)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5315..5337)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(5915..5920,5921..5936,5937..5943)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5951..5967
                     /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             5990..6643
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     6601..6623
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             6650..6673
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    join(6698..6736,1..86)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt
       61 aaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt
      121 atgcttccgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga ttttcaggag
      181 ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat
      241 ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga
      301 ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt
      361 atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg
      421 caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc
      481 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt
      541 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta
      601 aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt
      661 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat
      721 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt
      781 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
      841 agggcggggc gtaaagatct ggatccggct tactaaaagc cagataacag tatgcgtatt
      901 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
      961 aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     1021 gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa
     1081 gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc
     1141 gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca
     1201 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     1261 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     1321 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     1381 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     1441 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     1501 aatgtcaggc tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt
     1561 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     1621 ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggttgat gggtcgaggc
     1681 atcatcatca tcatcatcat catcatcact agtcgactcg agcggccgca tcgtgactga
     1741 ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     1801 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     1861 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     1921 ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1981 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2041 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2101 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2161 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2221 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2281 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2341 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     2401 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2461 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2521 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2581 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2641 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     2701 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2761 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2821 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2881 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2941 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3001 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3061 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3781 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3841 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3901 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3961 ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggcgc
     4021 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     4081 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     4141 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     4201 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     4261 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     4321 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     4381 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     4441 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     4501 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     4561 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     4621 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     4681 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     4741 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     4801 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     4861 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     4921 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     4981 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     5041 gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc
     5101 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     5161 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt
     5221 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5281 cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5341 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     5401 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5461 ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc
     5521 ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc
     5581 ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga
     5641 atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc
     5701 agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg
     5761 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg
     5821 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     5881 ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata
     5941 atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagtattca tgtcccctat
     6001 actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct
     6061 tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa
     6121 aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa
     6181 attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg
     6241 ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata
     6301 cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag
     6361 caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa
     6421 tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata
     6481 catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga
     6541 agctatccca caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca
     6601 gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggaagttct
     6661 gttccagggg cccctgggat ccccggaatt cccatcaaca agtttgtaca aaaaagctga
     6721 acgagaaacg taaaat
//
