LOCUS Exported 4896 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses mouse CAD (Irg1) in E.coli. ACCESSION . VERSION . KEYWORDS pCAD39_mIRG1_4-462_pvp008 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4896 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /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(4859..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 //