LOCUS       sequence-247193-        4956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Aspergillus terreus CAD in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4956)
  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 4956)
  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: pCAD16_CAD_12-490_pvp008
FEATURES             Location/Qualifiers
     source          1..4956
                     /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             1705..1749
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1787..1805)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1801..1848
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2078..2893)
                     /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(2078..2893)
                     /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     2801..2820
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2894..2985)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3003..3638)
                     /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(3148..3167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3836..4918)
                     /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     4880..4899
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(4919..4956,1..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 cagcggtgtt accagcgaaa tttgtcattg ggcaagcaat ctggcaaccg atgatattcc
      301 gagtgatgtt ctggaacgtg ccaaatatct gattctggat ggtattgcat gtgcatgggt
      361 tggtgcacgt gttccgtggt cagaaaaata tgttcaggca accatgagct ttgaaccgcc
      421 tggtgcatgt cgtgttattg gttatggcca gaaactgggt ccggttgcag cagcaatgac
      481 caatagcgca tttattcagg ccaccgaact ggatgattat catagcgaag caccgctgca
      541 tagcgcaagc attgttctgc ctgcagtttt tgcagcaagc gaagttctgg cagaacaggg
      601 taaaaccatt agcggtattg atgttattct ggcagccatt gttggttttg aaagcggtcc
      661 gcgtattggt aaagcaattt atggtagcga tctgctgaat aatggttggc attgtggtgc
      721 agtttatggt gcaccggcag gcgcactggc caccggtaaa ctgctgggtc tgacaccgga
      781 tagcatggaa gatgcactgg gtattgcctg tacccaggca tgtggtctga tgagcgcaca
      841 gtatggtggt atggttaaac gtgttcagca tggttttgca gcccgtaatg gtctgctggg
      901 tggcctgctg gcacatggtg gttatgaagc aatgaaaggt gtgctggaac gtagctatgg
      961 tggttttctg aaaatgttta ccaaaggcaa tggtcgtgaa cctccgtata aagaagaaga
     1021 agttgttgca ggtctgggta gcttttggca tacctttacc attcgcatta aactgtatgc
     1081 atgttgtggt ctggttcatg gtccggtgga agcaattgaa aatctgcagg gtcgttatcc
     1141 ggaactgctg aatcgtgcaa atctgagcaa tattcgtcat gttcatgttc agctgagcac
     1201 cgcaagcaat agccattgcg gttggattcc ggaagaacgt ccgattagca gcattgcagg
     1261 tcagatgagc gttgcatata ttctggccgt tcagctggtt gatcagcagt gtctgctgag
     1321 ccagtttagc gaatttgatg ataacctgga acgtccggaa gtttgggatc tggcacgtaa
     1381 agttaccagc agccagagcg aagaatttga tcaggatggt aattgtctga gcgcaggtcg
     1441 tgttcgtatt gaatttaatg atggttccag cattaccgaa agcgttgaaa aaccgctggg
     1501 tgttaaagaa ccgatgccga atgaacgtat cctgcataaa tatcgtaccc tggcaggtag
     1561 cgttaccgat gaaagccgtg tgaaagaaat tgaagatctg gttctgggtc tggatcgtct
     1621 gaccgatatt agtccgctgc tggaactgct gaactgtccg gttaaaagtc cgctggtttg
     1681 aagcttgcat gcctcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac
     1741 atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa
     1801 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag
     1861 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     1921 tagacataag cggctattta acgaccctgc cctgaaccga cgacaagctg acgaccgggt
     1981 ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2041 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     2101 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     2161 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2221 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2281 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2341 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2401 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2461 ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2521 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2581 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2641 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2701 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2761 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2821 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2881 gaatatggct catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2941 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggc atgctagcgc
     3001 agaaacgtcc tagaagatgc caggaggata cttagcagag agacaataag gccggagcga
     3061 agccgttttt ccataggctc cgcccccctg acgaacatca cgaaatctga cgctcaaatc
     3121 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct gatggctccc
     3181 tcttgcgctc tcctgttccc gtcctgcggc gtccgtgttg tggtggaggc tttacccaaa
     3241 tcaccacgtc ccgttccgtg tagacagttc gctccaagct gggctgtgtg caagaacccc
     3301 ccgttcagcc cgactgctgc gccttatccg gtaactatca tcttgagtcc aacccggaaa
     3361 gacacgacaa aacgccactg gcagcagcca ttggtaactg agaattagtg gatttagata
     3421 tcgagagtct tgaagtggtg gcctaacaga ggctacactg aaaggacagt atttggtatc
     3481 tgcgctccac taaagccagt taccaggtta agcagttccc caactgactt aaccttcgat
     3541 caaaccgcct ccccaggcgg ttttttcgtt tacagagcag gagattacga cgatcgtaaa
     3601 aggatctcaa gaagatcctt tacggattcc cgacaccatc actctagatt tcagtgcaat
     3661 ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat
     3721 gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     3781 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     3841 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3901 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     3961 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4021 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4081 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     4141 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4201 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4261 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4321 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4381 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     4441 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     4501 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     4561 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     4621 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     4681 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     4741 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     4801 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     4861 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     4921 tcaccaccct gaattgactc tcttccgggc gctatc
//
