LOCUS       sequence-411047-        4378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of R196E wgMDH with IPTG induction - Active 
            site mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4378)
  AUTHORS   Cox B, Chit MM, Weaver T, Gietl C, Bailey J, Bell E, Banaszak L
  TITLE     Organelle and translocatable forms of glyoxysomal malate 
            dehydrogenase. The effect of the N-terminal presequence.
  JOURNAL   FEBS J. 2005 Feb;272(3):643-54. doi: 
            10.1111/j.1742-4658.2004.04475.x.
  PUBMED    15670147
REFERENCE   2  (bases 1 to 4378)
  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"; doi: 
            "10.1111/j.1742-4658.2004.04475"; journalName: "FEBS J"; date: 
            "2005-02"; volume: "272"; issue: "3"; pages: "643-54"
COMMENT     Sequence Label: pQE60 R196E wgMDH
FEATURES             Location/Qualifiers
     source          1..4378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             566..577
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             1081..1098
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1121..1215
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1325..1344)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1987..2073
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2120..2142)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2228..2368
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2283..2302
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2383..2400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2554..3142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2634..2653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3313..4104,4105..4173))
                     /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     3936..3955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4174..4278)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4346..4364
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatggct
      121 aaaggcggag ctcccgggtt caaagtcgca atacttggcg ctgccggtgg cattggccag
      181 ccccttgcga tgttgatgaa gatgaatcct ctggtttctg ttctacatct atatgatgta
      241 gtcaatgccc ctggtgtcac cgctgatatt agccacatgg acacgggtgc tgtggtgcgt
      301 ggattcttgg ggcagcagca gctggaggct gcgcttactg gcatggatct tattatagtc
      361 cctgcaggtg ttcctcgaaa accaggaatg acgagggatg atctgttcaa aataaacgca
      421 ggaattgtca agactctgtg tgaagggatt gcaaagtgtt gtccaagagc cattgtcaac
      481 ctgatcagta atcctgtgaa ctccaccgtg cccatcgcag ctgaagtttt caagaaggct
      541 ggaacttatg atccaaagcg acttctggga gttacaatgc tcgacgtagt cgaagccaat
      601 acctttgtgg cagaagtatt gggtcttgat cctcgggatg ttgatgttcc agttgttggc
      661 ggtcatgctg gtgtaaccat tttgcccctt ctatctcagg tgaagcctcc aagttctttc
      721 acacaagaag agattagtta cctgactgat aggattcaaa atggtggaac agaagttgtc
      781 gaggccaaag caggagctgg ttcagcaact ctctcaatgg cttatgctgc cgttaagttt
      841 gcagatgcat gcctcagggg cttaagagga gatgctggtg tcattgaatg cgcgtttgtg
      901 tcttctcagg tgactgaact tccattcttt gcatcaaaag tacgacttgg tcgcaatggt
      961 atcgaagaag tatactccct tggcccgcta aatgagtatg agaggattgg attggagaaa
     1021 gcgaagaaag agttggcagg aagcattgag aagggagttt ccttcatcag aagcagatct
     1081 catcaccatc accatcacta agcttaatta gctgagcttg gactcctgtt gatagatcca
     1141 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     1201 tttattggtg agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat
     1261 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     1321 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     1381 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     1441 cattcttgcc cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga
     1501 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     1561 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     1621 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     1681 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     1741 caatatggac aacttcttcg cccccgtttt caccatgcat gggcaaatat tatacgcaag
     1801 gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc
     1861 atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt
     1921 aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact ctctagcttg
     1981 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2041 ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagagctgc ctcgcgcgtt
     2101 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2161 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2221 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     2281 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     2341 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     2401 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2461 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     3001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3421 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3541 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3601 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3721 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3841 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3961 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4021 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4141 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4201 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4261 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     4321 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcac
//
