LOCUS       sequence-238504-        4893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hCAD His103Ala mutant in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4893)
  AUTHORS   Chen F, Lukat P, Iqbal AA, Saile K, Kaever V, van den Heuvel J, 
            Blankenfeldt W, Bussow K, Pessler F
  TITLE     Crystal structure of cis-aconitate decarboxylase reveals the impact 
            of naturally occurring human mutations on itaconate synthesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 1908770116. doi: 
            10.1073/pnas.1908770116.
  PUBMED    31548418
REFERENCE   2  (bases 1 to 4893)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Sep 23. pii: 1908770116. doi: 
            10.1073/pnas.1908770116."
COMMENT     Sequence Label: pCAD54_CAD29_H104A_pvp008
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     86..105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        86..104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    105..129
                     /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             182..205
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             212..232
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1642..1686
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1724..1742)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1738..1785
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2015..2830)
                     /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(2015..2830)
                     /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     2738..2757
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2831..2922)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2940..3575)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(3085..3104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3773..4855)
                     /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     4817..4836
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(4856..4893,1..40))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
       61 ttatgcgact cctgcattag gaaattaata cgactcacta taggggaatt gtgagcggat
      121 aacaattccc ctgtagaaat aattttgttt aactttaata aggagatata ccatggctag
      181 ctggagccac ccgcagttcg aaaaagtcga cgagaacctc tacttccagg gaggcggccg
      241 caagtctatc acagaaagct ttgccacagc aatccatggc ttgaaagtgg gacacctgac
      301 agatcgtgtt attcagagga gcaagaggat gattctagac actctgggtg ctgggttcct
      361 gggaaccact acggaagtgt ttcacatagc cagccaatat agcaagatct acagttccaa
      421 catatccagc actgtttggg gtcagccaga catcaggctc ccgcccacat atgctgcttt
      481 tgtgaacggt gtggctattc actccatgga ttttgatgac acgtggcacc ctgccaccgc
      541 cccttctggg gctgtccttc ctgtcctcac agctttagca gaagccctgc caaggagtcc
      601 aaagttttct ggccttgacc tgctgctggc tttcaatgtt ggtattgaag tgcaaggccg
      661 attactgcat ttcgccaagg aggccaatga catgccaaag agattccatc ccccttccgt
      721 ggtaggaacg ttgggtagtg ctgctgctgc atccaagttt ttaggactta gctcgacaaa
      781 gtgccgagaa gctctggcca ttgctgtttc ccatgctggg gcacccatgg ccaatgctgc
      841 cacccagacc aagcccctcc acattggcaa tgctgccaag catgggatag aagctgcatt
      901 tttggcaatg ttgggtctcc aaggaaacaa gcaggtcttg gacttggagg caggatttgg
      961 ggccttttat gccaactatt ccccaaaagt ccttccaagc atagcttcct acagttggct
     1021 gctggaccag caggacgtgg cctttaagcg ttttcctgca catttatcta cccactgggt
     1081 ggcagacgca gctgcatctg tgagaaagca ccttgtagca gagagagccc tgcttccaac
     1141 tgactacatt aagagaattg tgctcaggat accaaatgtc cagtatgtaa acaggccctt
     1201 tccagtttcg gagcatgaag cccgtcattc attccagtat gtggcctgtg ccatgctgct
     1261 tgatggtggc atcactgtcc cctcattcca tgaatgccag atcaacaggc cacaggtgag
     1321 agagctgctc agtaaggtgg agctggagta ccctccggac aacttgccaa gcttcaacat
     1381 actgtactgt gaaataagtg tcaccctcaa ggatggagcc accttcacag atcgctctga
     1441 taccttctat gggcactgga gaaaaccact gagccaggag gacctagagg aaaagttcag
     1501 agccaatgcc tccaagatgc tgtcctggga cacagtggaa agccttataa agatagtcaa
     1561 aaatctagaa gacctagaag actgttctgt gttaactaca cttctcaaag gaccctgaag
     1621 cttgcatgcc tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
     1681 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
     1741 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca
     1801 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
     1861 acataagcgg ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc
     1921 cgcaagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1981 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2041 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2101 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2161 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2221 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2281 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2341 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt
     2401 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2461 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2521 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2581 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2641 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2701 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     2761 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     2821 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     2881 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg ctagcgcaga
     2941 aacgtcctag aagatgccag gaggatactt agcagagaga caataaggcc ggagcgaagc
     3001 cgtttttcca taggctccgc ccccctgacg aacatcacga aatctgacgc tcaaatcagt
     3061 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgat ggctccctct
     3121 tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg tggaggcttt acccaaatca
     3181 ccacgtcccg ttccgtgtag acagttcgct ccaagctggg ctgtgtgcaa gaaccccccg
     3241 ttcagcccga ctgctgcgcc ttatccggta actatcatct tgagtccaac ccggaaagac
     3301 acgacaaaac gccactggca gcagccattg gtaactgaga attagtggat ttagatatcg
     3361 agagtcttga agtggtggcc taacagaggc tacactgaaa ggacagtatt tggtatctgc
     3421 gctccactaa agccagttac caggttaagc agttccccaa ctgacttaac cttcgatcaa
     3481 accgcctccc caggcggttt tttcgtttac agagcaggag attacgacga tcgtaaaagg
     3541 atctcaagaa gatcctttac ggattcccga caccatcact ctagatttca gtgcaattta
     3601 tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt
     3661 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     3721 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     3781 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3841 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     3901 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     3961 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4021 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     4081 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4141 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4201 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4261 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4321 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4381 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4441 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4501 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4561 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     4621 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     4681 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     4741 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     4801 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     4861 ccaccctgaa ttgactctct tccgggcgct atc
//
