LOCUS       sequence-257458-        6274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hCAD Arg273His mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6274)
  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 6274)
  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: pCAD108_Arg273His_pCMV6Entry-hIRG1
FEATURES             Location/Qualifiers
     source          1..6274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     165..182
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     952..971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2440..2469
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2488..2511
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2531..2547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2531..2547)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2558..3180
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2757..2776)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(3329..3917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3409..3428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4246..4293
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4318..4337
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4525..5319)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4525..5319)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4636..4655)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5246..5265
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5354..5711)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5368..5503
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5520..5711
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5668..5688
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(5713..5817)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5844..6272
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5931..5950)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6141..6162
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccggga attcatgatg ctcaagtcta tcacagaaag ctttgccaca
     1021 gcaatccatg gcttgaaagt gggacacctg acagatcgtg ttattcagag gagcaagagg
     1081 atgattctag acactctggg tgctgggttc ctgggaacca ctacggaagt gtttcacata
     1141 gccagccaat atagcaagat ctacagttcc aacatatcca gcactgtttg gggtcagcca
     1201 gacatcaggc tcccgcccac atatgctgct tttgtgaacg gtgtggctat tcactccatg
     1261 gattttgatg acacgtggca ccctgccacc cacccttctg gggctgtcct tcctgtcctc
     1321 acagctttag cagaagccct gccaaggagt ccaaagtttt ctggccttga cctgctgctg
     1381 gctttcaatg ttggtattga agtgcaaggc cgattactgc atttcgccaa ggaggccaat
     1441 gacatgccaa agagattcca tcccccttcc gtggtaggaa cgttgggtag tgctgctgct
     1501 gcatccaagt ttttaggact tagctcgaca aagtgccgag aagctctggc cattgctgtt
     1561 tcccatgctg gggcacccat ggccaatgct gccacccaga ccaagcccct ccacattggc
     1621 aatgctgcca agcatgggat agaagctgca tttttggcaa tgttgggtct ccaaggaaac
     1681 aagcaggtct tggacttgga ggcaggattt ggggcctttt atgccaacta ttccccaaaa
     1741 gtccttccaa gcatagcttc ctacagttgg ctgctggacc agcaggacgt ggcctttaag
     1801 cattttcctg cacatttatc tacccactgg gtggcagacg cagctgcatc tgtgagaaag
     1861 caccttgtag cagagagagc cctgcttcca actgactaca ttaagagaat tgtgctcagg
     1921 ataccaaatg tccagtatgt aaacaggccc tttccagttt cggagcatga agcccgtcat
     1981 tcattccagt atgtggcctg tgccatgctg cttgatggtg gcatcactgt cccctcattc
     2041 catgaatgcc agatcaacag gccacaggtg agagagctgc tcagtaaggt ggagctggag
     2101 taccctccgg acaacttgcc aagcttcaac atactgtact gtgaaataag tgtcaccctc
     2161 aaggatggag ccaccttcac agatcgctct gataccttct atgggcactg gagaaaacca
     2221 ctgagccagg aggacctaga ggaaaagttc agagccaatg cctccaagat gctgtcctgg
     2281 gacacagtgg aaagccttat aaagatagtc aaaaatctag aagacctaga agactgttct
     2341 gtgttaacta cacttctcaa aggaccctct ccaccagagg tagcttcaaa ctctccagca
     2401 tgtaataatt ctatcacaaa tctctccaag cggccgctcg agcagaaact catctcagaa
     2461 gaggatctgg cagcaaatga tatcctggat tacaaggatg acgacgataa ggtttaaacg
     2521 gccggccgcg gtcatagctg tttcctgaac agatcccggg tggcatccct gtgacccctc
     2581 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     2641 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     2701 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     2761 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     2821 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     2881 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     2941 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     3001 acaggcgtga accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca
     3061 ggaggacgtc cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag
     3121 ttgcttgctt ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt
     3181 gggtacgcgg ccagcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3241 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3301 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3361 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3421 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3481 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3541 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3601 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3661 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3721 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3781 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3841 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3901 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3961 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4021 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taacctgagg
     4081 ctatggcagg gcctgccgcc ccgacgttgg ctgcgagccc tgggccttca cccgaacttg
     4141 gggggtgggg tggggaaaag gaagaaacgc gggcgtattg gccccaatgg ggtctcggtg
     4201 gggtatcgac agagtgccag ccctgggacc gaaccccgcg tttatgaaca aacgacccaa
     4261 caccgtgcgt tttattctgt ctttttattg ccgtcatagc gcgggttcct tccggtattg
     4321 tctccttccg tgtttcagtt agcctccccc tagggtgggc gaagaactcc agcatgagat
     4381 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     4441 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
     4501 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     4561 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     4621 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc gatccgccac
     4681 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     4741 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc tcgccttgag
     4801 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     4861 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     4921 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     4981 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     5041 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     5101 cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc agttcattca gggcaccgga
     5161 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     5221 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     5281 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     5341 ctcttgatcg atctttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     5401 atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     5461 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     5521 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     5581 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     5641 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agctggttct
     5701 ttccgcctca ggactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5761 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5821 atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5881 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5941 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     6001 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     6061 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     6121 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     6181 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     6241 tgagctgatt taacaaaaat ttaacgcgaa tttt
//
