LOCUS       sequence-459002-        6614 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For T-REX experiments of CYP2A6 C14A, C82A, and C439A triple mutant.
            Almost no sensing ability to reactive electrophilic species (RES)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6614)
  TITLE     Organ-specific electrophile responsivity mapping in live C. elegans
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6614)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCS2+8-CYP2A6(C14A, C82A, C439A)-flag-tev-halo...
FEATURES             Location/Qualifiers
     source          1..6614
                     /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             1681..2571
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             2581..2598
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2666..2800
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2690..2709)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2744..2763
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2913..2933)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2913..2931)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2952..2968)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2952..2968)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2965..2987)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2976..2992
                     /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(3000..3006,3007..3024,3025..3030))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3045..3066
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3183..3200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3354..3942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3434..3453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4113..4904,4905..4973))
                     /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     4736..4755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4974..5078)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5104..5559)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5241..5262)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5453..5472
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5630..6614
                     /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 ggtcgccctg
      181 actgtgatgg tcttgatgtc tgtttggcag cagaggaaga gcaaggggaa gctgcctccg
      241 ggacccaccc cattgccctt cattggaaac tacctgcaac tgaacacaga gcagatgtac
      301 aactccctca tgaagatcag tgagcgctat ggccccgtgt tcaccattca cttggggccc
      361 cggcgggtcg tggtgctggc 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 gggctcaggg
     1681 atggcagaaa tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag
     1741 cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt
     1801 aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc
     1861 tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc
     1921 ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc
     1981 gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca
     2041 gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa
     2101 tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag
     2161 ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg
     2221 ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag
     2281 ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg
     2341 ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg
     2401 ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct
     2461 aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac
     2521 ctgatcggca gcgagatcgc gcgctggctg tcgacgctcg agatttccgg cggcagcagc
     2581 catcatcatc atcatcatgg gtcagggtaa taatcgaatt caaggcctct cgagcctcta
     2641 gaactatagt gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg
     2701 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     2761 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     2821 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc
     2881 aatgcattgg gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     2941 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3001 aacatacgag ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3061 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3121 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3181 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3241 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3301 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3361 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3421 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3481 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3541 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3601 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3661 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3721 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3781 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3841 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3901 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3961 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4021 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4081 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4141 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4201 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4261 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4321 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4381 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4441 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4501 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4561 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4621 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4681 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4741 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4801 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4861 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4921 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4981 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5041 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5101 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     5161 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     5221 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     5281 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     5341 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     5401 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     5461 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     5521 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     5581 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag tcgaccgcca
     5641 attcaatatg gcgtatatgg actcatgcca attcaatatg gtggatctgg acctgtgcca
     5701 attcaatatg gcgtatatgg actcgtgcca attcaatatg gtggatctgg accccagcca
     5761 attcaatatg gcggacttgg caccatgcca attcaatatg gcggacctgg cactgtgcca
     5821 actggggagg ggtctacttg gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc
     5881 aagtccgcca tattgaattg gcatggtgcc aataatggcg gccatattgg ctatatgcca
     5941 ggatcaatat ataggcaata tccaatatgg ccctatgcca atatggctat tggccaggtt
     6001 caatactatg tattggccct atgccatata gtattccata tatgggtttt cctattgacg
     6061 tagatagccc ctcccaatgg gcggtcccat ataccatata tggggcttcc taataccgcc
     6121 catagccact cccccattga cgtcaatggt ctctatatat ggtctttcct attgacgtca
     6181 tatgggcggt cctattgacg tatatggcgc ctcccccatt gacgtcaatt acggtaaatg
     6241 gcccgcctgg ctcaatgccc attgacgtca ataggaccac ccaccattga cgtcaatggg
     6301 atggctcatt gcccattcat atccgttctc acgcccccta ttgacgtcaa tgacggtaaa
     6361 tggcccactt ggcagtacat caatatctat taatagtaac ttggcaagta cattactatt
     6421 ggaagtacgc cagggtacat tggcagtact cccattgacg tcaatggcgg taaatggccc
     6481 gcgatggctg ccaagtacat ccccattgac gtcaatgggg aggggcaatg acgcaaatgg
     6541 gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg
     6601 ctcgtttagg gaac
//
