LOCUS       sequence-459057-        5717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For overexpression of CYP2A6 C439A mutant in mammalian cells and 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5717)
  AUTHORS   Liu J, Kulkarni A, Gao YQ, Urul DA, Hamelin R, Novotny BA, Long MJC,
            Aye Y
  TITLE     Organ-specific electrophile responsivity mapping in live C. elegans.
  JOURNAL   Cell. 2024 Dec 26;187(26):7450-7469.e29. doi: 
            10.1016/j.cell.2024.10.014. Epub 2024 Nov 5.
  PUBMED    39504959
REFERENCE   2  (bases 1 to 5717)
  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"; doi: 
            "10.1016/j.cell.2024.10.014"; journalName: "Cell"; date: 
            "2024-12-26- 26"; volume: "187"; issue: "26"; pages: "7450-7469.e29"
COMMENT     Sequence Label: pCS2+8-CYP2A6(C439A)-flag-tev-his
FEATURES             Location/Qualifiers
     source          1..5717
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1624..1647
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1654..1674
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1684..1701
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1769..1903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1793..1812)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1847..1866
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2016..2036)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2016..2034)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2055..2071)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2055..2071)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2068..2090)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2079..2095
                     /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)."
     promoter        complement(join(2103..2109,2110..2127,2128..2133))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2148..2169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2286..2303)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2457..3045)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2537..2556)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3216..4007,4008..4076))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3839..3858
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4077..4181)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4207..4662)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4344..4365)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4556..4575
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4733..5717
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccactagt ggcgcgccat taattaaagg ccggccagcg
      121 atcgccggac ccaccatgct ggcctcaggg atgcttctgg tggccttgct ggtctgcctg
      181 actgtgatgg tcttgatgtc tgtttggcag cagaggaaga gcaaggggaa gctgcctccg
      241 ggacccaccc cattgccctt cattggaaac tacctgcaac tgaacacaga gcagatgtac
      301 aactccctca tgaagatcag tgagcgctat ggccccgtgt tcaccattca cttggggccc
      361 cggcgggtcg tggtgctgtg tggacatgat gccgtcaggg aggctctggt ggaccaggct
      421 gaggagttca gcgggcgagg cgagcaagcc accttcgact gggtcttcaa aggctatggc
      481 gtggtattca gcaacgggga gcgcgccaag cagctccggc gcttctccat cgccaccctg
      541 cgggacttcg gggtgggcaa gcgaggcatc gaggagcgca tccaggagga ggcgggcttc
      601 ctcatcgacg ccctccgggg cactggcggc gccaatatcg atcccacctt cttcctgagc
      661 cgcacagtct ccaatgtcat cagctccatt gtctttgggg accgctttga ctataaggac
      721 aaagagttcc tgtcactgtt gcgcatgatg ctaggaatct tccagttcac gtcaacctcc
      781 acggggcagc tctatgagat gttctcttcg gtgatgaaac acctgccagg accacagcaa
      841 caggcctttc agttgctgca agggctggag gacttcatag ccaagaaggt ggagcacaac
      901 cagcgtacgc tggatcccaa ttccccacgg gacttcattg actcctttct catccgcatg
      961 caggaggagg agaagaaccc caacacggag ttctacttga aaaacctggt gatgaccacg
     1021 ttgaacctct tcattggggg caccgagacc gtcagcacca ccctgcgcta tggcttcttg
     1081 ctgctcatga agcacccaga ggtggaggcc aaggtccatg aggagattga cagagtgatc
     1141 ggcaagaacc ggcagcccaa gtttgaggac cgggccaaga tgccctacat ggaggcagtg
     1201 atccacgaga tccaaagatt tggagacgtg atccccatga gtttggcccg cagagtcaaa
     1261 aaggacacca agtttcggga tttcttcctc cctaagggca ccgaagtgta ccctatgctg
     1321 ggctctgtgc tgagagaccc cagtttcttc tccaaccccc aggacttcaa tccccagcat
     1381 ttcctgaatg agaaggggca gtttaagaag agtgatgctt ttgtgccctt ttccatcgga
     1441 aagcggaacg ctttcggaga aggcctggcc agaatggagc tctttctctt cttcaccacc
     1501 gtcatgcaga acttccgcct caagtcctcc cagtcaccta aggacattga cgtgtccccc
     1561 aaacacgtgg gctttgccac gatcccacga aactacacca tgagcttcct gccccgctca
     1621 ggggattata aagacgatga cgacaagtca ggggaaaact tgtatttcca gggcggcagc
     1681 agccatcatc atcatcatca tgggtcaggg taataatcga attcaaggcc tctcgagcct
     1741 ctagaactat agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt
     1801 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     1861 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     1921 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc
     1981 gccaatgcat tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc
     2041 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2101 cacaacatac gagccgggag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2161 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2221 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2281 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2341 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2401 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2461 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2521 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2581 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2641 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2701 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2761 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2821 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2881 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2941 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3001 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3061 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3121 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3181 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3241 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3301 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3361 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3421 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3481 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3541 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3601 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3661 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3721 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3781 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3841 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3901 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3961 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4021 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4081 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4141 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4201 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4261 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4321 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4381 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4441 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4501 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4561 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4621 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     4681 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgaccg
     4741 ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc tggacctgtg
     4801 ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc tggaccccag
     4861 ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggacc tggcactgtg
     4921 ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc ttggccctgt
     4981 gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat tggctatatg
     5041 ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc tattggccag
     5101 gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt tttcctattg
     5161 acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct tcctaatacc
     5221 gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt cctattgacg
     5281 tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca attacggtaa
     5341 atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat tgacgtcaat
     5401 gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt caatgacggt
     5461 aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa gtacattact
     5521 attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg cggtaaatgg
     5581 cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca atgacgcaaa
     5641 tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc tatataagca
     5701 atgctcgttt agggaac
//
