LOCUS       sequence-212030-        5117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and GST fusions with optional C-terminal 
            StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5117)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5117)
  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            "unpublished"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: pExp-GST
FEATURES             Location/Qualifiers
     source          1..5117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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)."
     RBS             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             131..781
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     739..761
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             834..857
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(871..889)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(872..889)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(947..965)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      961..1008
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1244..1265)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1415..2497)
                     /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     2459..2478
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2498..2575)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2714..2733
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2887..2906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3006..3028
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3066..3084)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3152..3256
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3257..3325,3326..4117)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3475..3494)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4288..4876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4777..4796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5030..5047
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg
      181 acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg
      241 tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta
      301 tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa
      361 gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc
      421 ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct
      481 caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg
      541 tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc
      601 tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg
      661 ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta
      721 tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa
      781 aagcggtacc gaaaacctgt acttccagtg agaccttaat taactcgagc gcatggagcc
      841 acccgcagtt cgaaaaataa gcttgagtat tctatagtgt cacctaaatc ccagcttgat
      901 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      961 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1021 actatatccg gataacctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     1081 agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc
     1141 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     1201 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     1261 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     1321 tggcccacag cttgcatgcc tgcaggtcgg aagcataaag tgtaaagcct ggggtgccta
     1381 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1441 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1501 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     1561 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     1621 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     1681 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     1741 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     1801 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     1861 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     1921 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     1981 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     2041 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     2101 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     2161 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     2221 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     2281 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     2341 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     2401 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     2461 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     2521 cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg gtgtcggcga
     2581 gctcgaattg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2641 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2701 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2761 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2821 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     2881 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     2941 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3001 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     3061 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3121 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3181 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3241 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3301 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3361 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3421 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     3481 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     3541 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     3601 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     3661 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     3721 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     3781 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     3841 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     3901 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     3961 cggtgagcgt ggttctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4021 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4081 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4141 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4201 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4261 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4321 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4381 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4441 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4501 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4561 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4621 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4681 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4741 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4801 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4861 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4921 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4981 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5041 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     5101 tcattaatgc agctgat
//
