LOCUS       sequence-328775-        6511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for His6-MEK1C218C222.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6511)
  AUTHORS   Lai KY, Galan SRG, Zeng Y, Zhou TH, He C, Raj R, Riedl J, Liu S, 
            Chooi KP, Garg N, Zeng M, Jones LH, Hutchings GJ, Mohammed S, Nair 
            SK, Chen J, Davis BG, van der Donk WA
  TITLE     LanCLs add glutathione to dehydroamino acids generated at 
            phosphorylated sites in the proteome.
  JOURNAL   Cell. 2021 Apr 27. pii: S0092-8674(21)00436-0. doi: 
            10.1016/j.cell.2021.04.001.
  PUBMED    33932340
REFERENCE   2  (bases 1 to 6511)
  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: "Cell. 2021 
            Apr 27. pii: S0092-8674(21)00436-0. doi: 
            10.1016/j.cell.2021.04.001."
COMMENT     Sequence Label: MEK1C218C222
FEATURES             Location/Qualifiers
     source          1..6511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        32..50
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    51..75
                     /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)."
     RBS             90..112
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             131..148
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             158..175
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1407..1424
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1477..1495)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1491..1538
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1575..2030
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1662..1681)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1872..1893
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2122..2937)
                     /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(2122..2937)
                     /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     2845..2864
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2929..2948
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3059..3647
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3548..3567
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3801..3818
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3833..3972
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3898..3917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4058..4080
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4074..4265)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5074..6156)
                     /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     6118..6137
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6157..6234)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6440..6459
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
       61 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat
      121 gttcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat
      181 gccgaaaaaa aaaccgaccc cgattcaact gaacccggct ccggatggct ccgctgtcaa
      241 tggcacctca agcgcagaaa cgaacctgga agcgctgcag aaaaaactgg aagaactgga
      301 actggatgaa cagcaacgta aacgcctgga agcctttctg acccagaaac aaaaagtggg
      361 cgaactgaaa gatgacgatt tcgaaaaaat cagtgaactg ggcgccggta acggcggtgt
      421 ggtttttaaa gtcagccata aaccgtctgg tctggtgatg gcacgtaaac tgattcacct
      481 ggaaatcaaa ccggctattc gtaaccagat tatccgcgaa ctgcaagtgc tgcatgaatg
      541 caattctccg tatattgttg gcttttatgg tgcgttctac agtgacggcg aaatttccat
      601 ctgtatggaa cacatggacg gcggtagcct ggatcaggtt ctgaaaaaag caggccgtat
      661 cccggaacaa attctgggta aagtctctat tgctgtgatc aaaggcctga cgtacctgcg
      721 tgaaaaacat aaaatcatgc accgcgatgt gaaaccgtca aacatcctgg ttaattcgcg
      781 cggtgaaatt aaactgtgcg actttggcgt tagcggtcag ctgattgatt gtatggcgaa
      841 ctgcttcgtc ggcacccgta gttatatgtc cccggaacgc ctgcagggta cgcattactc
      901 agtgcaatcg gatatctggt caatgggcct gtcgctggtt gaaatggccg ttggtcgtta
      961 tccgattccg ccgccggacg ccaaagaact ggaactgatg tttggctctc aggttgaagg
     1021 tgatgcggcc gaaaccccgc cgcgtccgcg taccccgggt cgtccgctga gctcttatgg
     1081 tatggacagc cgtccgccga tggcaatctt cgaactgctg gattacattg tgaatgaacc
     1141 gccgccaaaa ctgccgagcg gcgtgtttag cctggaattt caggacttcg tcaacaaatg
     1201 tctgatcaaa aatccggcag aacgtgctga tctgaaacaa ctgatggtcc acgcttttat
     1261 taaacgctcc gacgcggaag aagtggattt cgccggttgg ctgtcttcga ccattggcct
     1321 gaatcaaccg tcaacgccga cccacgctgc tggtgtgtaa ggatccgaat tcgagctccg
     1381 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     1441 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1501 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1561 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1621 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1681 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1741 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1801 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1861 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1921 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1981 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2041 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2101 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2161 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2221 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2281 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2341 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2401 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2461 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2521 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2581 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2641 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2701 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2761 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2821 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     2881 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     2941 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3001 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3061 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3121 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3181 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3241 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3301 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3361 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3421 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3481 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3541 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3601 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3661 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3721 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3781 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3841 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3901 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3961 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4021 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4081 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4141 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4201 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4261 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4321 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4381 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4441 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4501 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4561 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4621 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4681 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4741 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4801 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4861 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4921 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4981 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5041 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5101 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5161 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5221 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5281 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5341 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5401 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5461 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5521 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5581 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5641 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5701 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5761 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5821 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5881 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5941 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6001 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     6061 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     6121 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6181 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6241 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6301 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6361 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6421 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6481 cgccggtgat gccggccacg atgcgtccgg c
//
