LOCUS       Exported                6511 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression plasmid for His6-MEK1C222.
ACCESSION   .
VERSION     .
KEYWORDS    MEK1C222
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6511
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     9..28
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9..27
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    28..52
                     /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             67..89
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             108..125
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             135..152
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1384..1401
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1454..1472)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1468..1515
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1552..2007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1639..1658)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1849..1870
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2099..2914)
                     /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     2822..2841
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2906..2925
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3036..3624
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3525..3544
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3778..3795
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3810..3949
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3875..3894)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4035..4057
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4051..4242)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5051..6133)
                     /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     6095..6114
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6134..6211)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6417..6436
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
       61 aataattttg tttaacttta agaaggagat ataccatgtt cagcagccat catcatcatc
      121 atcacagcag cggcctggtg ccgcgcggca gccatatgcc gaaaaaaaaa ccgaccccga
      181 ttcaactgaa cccggctccg gatggctccg ctgtcaatgg cacctcaagc gcagaaacga
      241 acctggaagc gctgcagaaa aaactggaag aactggaact ggatgaacag caacgtaaac
      301 gcctggaagc ctttctgacc cagaaacaaa aagtgggcga actgaaagat gacgatttcg
      361 aaaaaatcag tgaactgggc gccggtaacg gcggtgtggt ttttaaagtc agccataaac
      421 cgtctggtct ggtgatggca cgtaaactga ttcacctgga aatcaaaccg gctattcgta
      481 accagattat ccgcgaactg caagtgctgc atgaatgcaa ttctccgtat attgttggct
      541 tttatggtgc gttctacagt gacggcgaaa tttccatctg tatggaacac atggacggcg
      601 gtagcctgga tcaggttctg aaaaaagcag gccgtatccc ggaacaaatt ctgggtaaag
      661 tctctattgc tgtgatcaaa ggcctgacgt acctgcgtga aaaacataaa atcatgcacc
      721 gcgatgtgaa accgtcaaac atcctggtta attcgcgcgg tgaaattaaa ctgtgcgact
      781 ttggcgttag cggtcagctg attgatagta tggcgaactg cttcgtcggc acccgtagtt
      841 atatgtcccc ggaacgcctg cagggtacgc attactcagt gcaatcggat atctggtcaa
      901 tgggcctgtc gctggttgaa atggccgttg gtcgttatcc gattccgccg ccggacgcca
      961 aagaactgga actgatgttt ggctctcagg ttgaaggtga tgcggccgaa accccgccgc
     1021 gtccgcgtac cccgggtcgt ccgctgagct cttatggtat ggacagccgt ccgccgatgg
     1081 caatcttcga actgctggat tacattgtga atgaaccgcc gccaaaactg ccgagcggcg
     1141 tgtttagcct ggaatttcag gacttcgtca acaaatgtct gatcaaaaat ccggcagaac
     1201 gtgctgatct gaaacaactg atggtccacg cttttattaa acgctccgac gcggaagaag
     1261 tggatttcgc cggttggctg tcttcgacca ttggcctgaa tcaaccgtca acgccgaccc
     1321 acgctgctgg tgtgtaagga tccgaattcg agctccgtcg acaagcttgc ggccgcactc
     1381 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1441 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1501 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1561 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1621 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1681 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1741 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1801 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1861 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1921 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1981 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     2041 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     2101 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     2161 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     2221 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     2281 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     2341 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     2401 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     2461 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     2521 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     2581 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     2641 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2701 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2761 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2821 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2881 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2941 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     3001 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3061 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3121 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3181 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3241 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3301 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3361 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3421 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3481 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3541 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3601 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3661 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3721 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3781 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3841 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3901 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3961 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4021 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4081 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     4141 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4201 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4261 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4321 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4381 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4441 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4501 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4561 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4621 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4681 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4741 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4801 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4861 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4921 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4981 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5041 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5101 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5161 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     5221 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     5281 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     5341 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5401 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5461 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5521 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5581 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5641 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5701 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5761 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5821 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5881 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5941 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6001 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6061 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6121 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6181 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     6241 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     6301 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6361 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6421 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6481 cgtccggcgt agaggatcga gatctcgatc c
//
