LOCUS       sequence-206041-        5931 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for bacterial expression of a His-tagged V156E mutant of GST
            encoded by preferred codons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5931)
  AUTHORS   Josse L, Sampson CDD, Tuite MF, Howland K, von der Haar T
  TITLE     Codon-Dependent Translational Accuracy Controls Protein Quality in 
            Escherichia coli but not in Saccharomyces cerevisiae
  JOURNAL   bioRxiv 200006
REFERENCE   2  (bases 1 to 5931)
  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: "bioRxiv 
            200006"
COMMENT     Sequence Label: pTH858-gst-optec V156E
FEATURES             Location/Qualifiers
     source          1..5931
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     120..139
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     223..242
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        223..241
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    242..266
                     /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             281..303
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             316..339
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1018..1035
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1088..1106)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1102..1149
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1186..1641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1273..1292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1483..1504
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1733..2548)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1733..2548)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2456..2475
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2540..2559
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2670..3258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3159..3178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3412..3429
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3444..3583
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3509..3528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3669..3691
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3685..3876)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4685..5767)
                     /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     5729..5748
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5768..5845)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
       61 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
      121 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      181 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
      241 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
      301 agatatacca tgggccacca tcaccatcac catcaccata gcccgattct gggctattgg
      361 aaaattaaag gcctggtgca gccgacccgc ctgctgctgg aatatctgga agaaaaatat
      421 gaagaacatc tgtatgaacg cgatgaaggc gataaatggc gcaacaaaaa atttgaactg
      481 ggcctggaat ttccgaacct gccgtattat attgatggcg atgtgaaact gacccagagc
      541 atggcgatta ttcgctatat tgcggataaa cataacatgc tgggcggctg cccgaaagaa
      601 cgcgcggaaa ttagcatgct ggaaggcgcg gtgctggata ttcgctatgg cgtgagccgc
      661 attgcgtata gcaaagattt tgaaaccctg aaagtggatt ttctgagcaa actgccggaa
      721 atgctgaaaa tgtttgaaga tcgcctgtgc cataaaacct atctgaacgg cgatcatgaa
      781 acccatccgg attttatgct gtatgatgcg ctggatgtgg tgctgtatat ggatccgatg
      841 tgcctggatg cgtttccgaa actggtgtgc tttaaaaaac gcattgaagc gattccgcag
      901 attgataaat atctgaaaag cagcaaatat attgcgtggc cgctgcaggg ctggcaggcg
      961 acctttggcg gcggcgatca tccgccgaaa taataaaagc ttgcggccgc actcgagcac
     1021 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1081 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1141 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1201 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1261 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1321 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1381 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1441 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1501 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1561 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1621 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1681 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1741 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1801 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1861 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1921 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1981 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2041 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2101 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2161 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2221 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2281 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2341 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2401 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2461 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2521 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2581 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2641 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2701 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2761 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2821 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2881 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2941 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3001 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3061 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3121 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3181 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3241 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3301 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3361 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3421 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3481 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3541 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3601 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3661 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3721 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3781 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3841 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3901 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3961 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4021 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4081 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4141 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4201 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4261 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4321 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4381 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4441 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4501 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4561 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4621 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4681 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4741 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4801 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4861 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4921 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     4981 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5041 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5101 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5161 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5221 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5281 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5341 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5401 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5461 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5521 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5581 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5641 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5701 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5761 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5821 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5881 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc a
//
