LOCUS       sequence-238487-        4896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse CAD (Irg1) in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4896)
  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 4896)
  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: pCAD39_mIRG1_4-462_pvp008
FEATURES             Location/Qualifiers
     source          1..4896
                     /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             1645..1689
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1727..1745)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1741..1788
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2018..2833)
                     /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(2018..2833)
                     /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     2741..2760
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2834..2925)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2943..3578)
                     /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(3088..3107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3776..4858)
                     /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     4820..4839
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(4859..4896,1..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 caagtctgtc acagagagct ttgctggtat gattcacggc ttgaaagtga accacctgac
      301 agatggtatc attcggagga gcaagaggat gatcctggat tctctgggcg ttggcttcct
      361 ggggacaggc acagaagtgt tccataaagt cacccaatat agtaaaatct acagttccaa
      421 cacctccagc actgtttggg gtcgaccaga cttcaggctc ccaccgacat atgctgcttt
      481 tgttaatggt gttgctgttc actccatgga ttttgatgac acatggcacc ctgccaccca
      541 cccttctggg gctgtcctac ctgtcctcac agctctatcg gaagccctgc ctcagactcc
      601 caagttttct ggcctcgacc tgctgctggc gttcaacgtt ggtattgaag tacagggacg
      661 attaatgcac ttctccaagg aagccaaaga cataccaaag agattccacc ctccctctgt
      721 ggtggggact ctgggaagtg ctgctgctgc gtccaagttt ctggggctca gcttgacaaa
      781 gtgccgcgag gcattggcta ttgctgtttc ccacgcaggg gcacccatag cgaacgctgc
      841 cactcagact aagccccttc atattggcaa tgcagccaag catgggatgg aagccacgtt
      901 tctggcaatg ctgggcctcc aaggaaacaa acagatcttg gacctggggt cagggttcgg
      961 tgccttctat gccaactact cccccgaaga ccttccaagc ctggattctc acatctggct
     1021 gttggaccag caggatgtgg cctttaagag cttcccggca catctggcta cccactgggt
     1081 ggcagatgca gctgcagccg tgagaaagca ccttgtgaca ccagaaagag ccctgttccc
     1141 tgctgaccac atcgagagaa tcgtgctcag gatccctgac gtccagtacg taaacaggcc
     1201 cttcccggac tcagagcatg aagcccgtca ttctttccag tatgtggcct gtgcctcgct
     1261 gctcgacggt agcatcactg tcccatcctt ccacagccag caggtcaata ggcctcaggt
     1321 gagagagttg ctcaagaagg tgaagctgga gcatcctcct gacaacccgc caagcttcga
     1381 cacgctatac tgtgaaataa gcatcactct aaaggacggg accactttca ccgagcgctc
     1441 tgacaccttc tatggtcact ggaggaaacc actgagccag gaagatctgc gcaacaagtt
     1501 ccgagccaat gcctcaaaga tgctatgcag ggacacggtg gaaagcctta taacggtagt
     1561 agaaaagcta gaagacctag aagactgctc tgtgctaacc agacttctga aaggaccctg
     1621 aagcttgcat gcctcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac
     1681 atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa
     1741 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag
     1801 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     1861 tagacataag cggctattta acgaccctgc cctgaaccga cgacaagctg acgaccgggt
     1921 ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1981 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     2041 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     2101 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2161 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2221 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2281 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2341 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2401 ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2461 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2521 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2581 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2641 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2701 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2761 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2821 gaatatggct catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2881 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggc atgctagcgc
     2941 agaaacgtcc tagaagatgc caggaggata cttagcagag agacaataag gccggagcga
     3001 agccgttttt ccataggctc cgcccccctg acgaacatca cgaaatctga cgctcaaatc
     3061 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct gatggctccc
     3121 tcttgcgctc tcctgttccc gtcctgcggc gtccgtgttg tggtggaggc tttacccaaa
     3181 tcaccacgtc ccgttccgtg tagacagttc gctccaagct gggctgtgtg caagaacccc
     3241 ccgttcagcc cgactgctgc gccttatccg gtaactatca tcttgagtcc aacccggaaa
     3301 gacacgacaa aacgccactg gcagcagcca ttggtaactg agaattagtg gatttagata
     3361 tcgagagtct tgaagtggtg gcctaacaga ggctacactg aaaggacagt atttggtatc
     3421 tgcgctccac taaagccagt taccaggtta agcagttccc caactgactt aaccttcgat
     3481 caaaccgcct ccccaggcgg ttttttcgtt tacagagcag gagattacga cgatcgtaaa
     3541 aggatctcaa gaagatcctt tacggattcc cgacaccatc actctagatt tcagtgcaat
     3601 ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat
     3661 gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     3721 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     3781 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3841 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     3901 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     3961 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4021 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     4081 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4141 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4201 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4261 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4321 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4381 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     4441 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     4501 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     4561 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     4621 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     4681 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     4741 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     4801 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     4861 tcaccaccct gaattgactc tcttccgggc gctatc
//
