LOCUS       sequence-233663-        5014 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Allows cloning of gene of interest into bacterial expression vector 
            with PreScission Protease cleavable N-terminal GST tag, and a 
            retained N-terminal FLAG tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5014)
  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  JOURNAL   Unpublished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 5014)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pGEX6P1-N-FLAG
FEATURES             Location/Qualifiers
     source          1..5014
                     /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"
     CDS             769..792
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(882..904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1049..1067)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1135..1239
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1240..1308,1309..2100)
                     /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(1458..1477)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2271..2859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2760..2779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3013..3030
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3103..3180
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3181..4263
                     /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(3200..3219)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4312..4317,4318..4335,4336..4342)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4350..4366
                     /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     4374..4390
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4374..4390
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4386..4913
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4406..4423)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4406..4422)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4415..4437)
                     /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 tgggatccga ctacaaagac
      781 gatgacgaca agcgatcccc ggaattcccg ggtcgactcg agcggccgca tcgtgactga
      841 ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
      901 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
      961 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     1021 ggagtgtata attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1081 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1141 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1201 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1261 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1321 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     1381 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     1441 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     1501 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     1561 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     1621 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     1681 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     1741 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     1801 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     1861 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     1921 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     1981 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2041 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2101 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2161 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2221 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2281 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2341 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2401 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2461 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2521 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2581 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2641 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2701 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2761 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2821 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2881 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2941 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3001 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3061 ctccttacgc atctgtgcgg tatttcacac cgcataaatt ccgacaccat cgaatggcgc
     3121 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     3181 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     3241 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     3301 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     3361 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     3421 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     3481 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     3541 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     3601 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     3661 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     3721 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     3781 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     3841 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     3901 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     3961 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     4021 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     4081 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     4141 gtctcactgg tgaaaagaaa aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc
     4201 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4261 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg ctttacactt
     4321 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     4381 cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     4441 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     4501 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     4561 ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg gagtgcgatc
     4621 ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt tacgatgcgc
     4681 ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt cccacggaga
     4741 atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta caggaaggcc
     4801 agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg tgcaccaatg
     4861 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg
     4921 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     4981 ataacggttc tggcaaatat tctgaaatga gctg
//
