LOCUS       sequence-329217-        6259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for His6-Delta-MEK1C218C222.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6259)
  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 6259)
  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: Delta-1-60MEK1C218C222
FEATURES             Location/Qualifiers
     source          1..6259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..52
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        33..51
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    52..76
                     /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             1089..1106
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1156..1173
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1226..1244)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1240..1287
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1324..1779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1411..1430)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1621..1642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1871..2686)
                     /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(1871..2686)
                     /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     2594..2613
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2678..2697
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2808..3396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3297..3316
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3550..3567
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3582..3721
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3647..3666)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3807..3829
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3823..4014)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4823..5905)
                     /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     5867..5886
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5906..5983)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6189..6208
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
       61 agcggataac aattcccctc tagaaataat tttgtttaac cttaagaagg agatatacta
      121 tggaactgaa agatgacgat ttcgaaaaaa tcagtgaact gggcgccggt aacggcggtg
      181 tggtttttaa agtcagccat aaaccgtctg gtctggtgat ggcacgtaaa ctgattcacc
      241 tggaaatcaa accggctatt cgtaaccaga ttatccgcga actgcaagtg ctgcatgaat
      301 gcaattctcc gtatattgtt ggcttttatg gtgcgttcta cagtgacggc gaaatttcca
      361 tctgtatgga acacatggac ggcggtagcc tggatcaggt tctgaaaaaa gcaggccgta
      421 tcccggaaca aattctgggt aaagtctcta ttgctgtgat caaaggcctg acgtacctgc
      481 gtgaaaaaca taaaatcatg caccgcgatg tgaaaccgtc aaacatcctg gttaattcgc
      541 gcggtgaaat taaactgtgc gactttggcg ttagcggtca gctgattgat tgtatggcga
      601 actgcttcgt cggcacccgt agttatatgt ccccggaacg cctgcagggt acgcattact
      661 cagtgcaatc ggatatctgg tcaatgggcc tgtcgctggt tgaaatggcc gttggtcgtt
      721 atccgattcc gccgccggac gccaaagaac tggaactgat gtttggctct caggttgaag
      781 gtgatgcggc cgaaaccccg ccgcgtccgc gtaccccggg tcgtccgctg agctcttatg
      841 gtatggacag ccgtccgccg atggcaatct tcgaactgct ggattacatt gtgaatgaac
      901 cgccgccaaa actgccgagc ggcgtgttta gcctggaatt tcaggacttc gtcaacaaat
      961 gtctgatcaa aaatccggca gaacgtgctg atctgaaaca actgatggtc cacgctttta
     1021 ttaaacgctc cgacgcggaa gaagtggatt tcgccggttg gctgtcttcg accattggcc
     1081 tgaatgcgca ccatcatcac caccattgag gatccgaatt cgagctccgt cgacaagctt
     1141 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1201 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1261 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1321 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1381 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1441 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1501 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1561 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1621 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1681 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1741 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1801 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1861 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1921 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1981 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     2041 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2101 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2161 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2221 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2281 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2341 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2401 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2461 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2521 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2581 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2641 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2701 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2761 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2821 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2881 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2941 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3001 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3061 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3121 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3181 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3241 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3301 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3361 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3421 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3481 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3541 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3601 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3661 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3721 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3781 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3841 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3901 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3961 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4021 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4081 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4141 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4201 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4261 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4321 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4381 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4441 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4501 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4561 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4621 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4681 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4741 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4801 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4861 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4921 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4981 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5041 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5101 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5161 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5221 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5281 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5341 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5401 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5461 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5521 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5581 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5641 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5701 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5761 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5821 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5881 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5941 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6001 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6061 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6121 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6181 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6241 ccggccacga tgcgtccgg
//
