LOCUS       Exported                6001 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  N-terminal MBP tagged human MEK1 gene S218TAG for bacterial 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    MBP-MEK1 S218TAG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6001)
  AUTHORS   Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, 
            Isaacs FJ, Rinehart J
  TITLE     A flexible codon in genomically recoded Escherichia coli permits 
            programmable protein phosphorylation.
  JOURNAL   Nat Commun. 2015 Sep 9;6:8130. doi: 10.1038/ncomms9130.
  PUBMED    26350500
REFERENCE   2  (bases 1 to 6001)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6001
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(152..775)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     683..702
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    824..840
                     /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)."
     protein_bind    847..863
                     /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        872..945
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    872..890
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    897..915
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             955..966
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             979..1001
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1010..2110
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2084..2107
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2159..2170
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2171..2191
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3389..3406
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3502..3520)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3516..3563
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3660..4018
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3747..3766)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3957..3978
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4043..4147
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4148..5008
                     /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     complement(4366..4385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5179..5767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5668..5687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5921..5938
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctatgat atcgattttt atttgatgcc tggcagttcc ctactctcgc atggggagac
       61 cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
      121 accaccgcgc tactgccgcc aggcatctag attaagaccc actttcacat ttaagttgtt
      181 tttctaatcc gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt
      241 gatcaaataa ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt
      301 ccctttcttc tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat
      361 gccccacagc gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa
      421 aggctaattg attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta
      481 cttttgctcc atcgcgatga cttagtaaag cacatctaaa acttttagcg ttattacgta
      541 aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca
      601 tctcaatggc taaggcgtcg agcaaagccc gcttattttt tacatgccaa tacaatgtag
      661 gctgctctac acctagcttc tgggcgagtt tacgggttgt taaaccttcg attccgacct
      721 cattaagcag ctctaatgcg ctgttaatca ctttactttt atctaatctg gacatgtcga
      781 atatagacgt cggtcagtgc gtcctgctga tgtgctcagt atcttgttat ccgctcacaa
      841 tgtcaattgt tatccgctca caattctcga gtccctatca gtgatagaga ttgacatccc
      901 tatcagtgat agagatactg agcacatcag caggacgcac tgaccgaatt cattaaagag
      961 gagaaagcta gaaataattt tgtttaactt taagaaggag atatacataa tgaaaatcga
     1021 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
     1081 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
     1141 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
     1201 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
     1261 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
     1321 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaagg atctgctgcc
     1381 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
     1441 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
     1501 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
     1561 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
     1621 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
     1681 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
     1741 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
     1801 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
     1861 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     1921 gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa
     1981 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     2041 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     2101 cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat
     2161 cgagggaagg gaaaatctgt attttcaagg tcatatgccg aagaagaaac cgaccccgat
     2221 ccagctgaac ccggctccgg acggttctgc tgttaacggc acctcttctg ctgaaaccaa
     2281 cctggaagct ctgcaaaaga aactggaaga actggaactg gacgaacagc agcgtaaacg
     2341 tctggaagcg ttcctgaccc agaaacagaa agttggtgaa ctgaaagacg acgacttcga
     2401 aaaaatctct gaactgggtg ctggtaacgg tggtgttgtt ttcaaagttt ctcacaaacc
     2461 gtccggtctg gttatggctc gtaaactgat ccacctggaa atcaaaccgg ctatccgtaa
     2521 ccagatcatc cgtgaactgc aagttctgca cgaatgcaac tctccgtaca tcgttggttt
     2581 ctacggtgct ttctactctg acggtgaaat ctctatctgc atggaacaca tggacggtgg
     2641 ttctctggac caggttctga aaaaagctgg tcgtatcccg gaacagatcc tgggtaaagt
     2701 ttctatcgct gttatcaaag gtctgaccta cctgcgtgaa aaacacaaaa tcatgcaccg
     2761 tgacgttaaa ccgtctaaca tcctggttaa ctctcgtggt gaaatcaaac tgtgcgactt
     2821 cggtgtttct ggtcagctga tcgactagat ggctaactct ttcgttggca cccgttctta
     2881 catgtctccg gaacgtctgc aaggcaccca ctactctgtt cagtctgaca tctggtctat
     2941 gggtctgtct ctggttgaaa tggctgttgg tcgttacccg atcccgccgc cggacgctaa
     3001 agaactggaa ctgatgttcg gttgccaggt tgaaggtgac gctgctgaaa ccccgccgcg
     3061 tccgcgtact ccgggtcgtc cgctgtcttc ttacggtatg gactctcgtc cgccgatggc
     3121 tatcttcgaa ctgctggact acatcgttaa cgaaccgcct ccgaaactgc cgtctggtgt
     3181 tttctctctg gagttccagg acttcgttaa caaatgcctg atcaaaaacc cggctgaacg
     3241 tgctgacctg aaacagctga tggttcacgc tttcatcaaa cgttctgacg ctgaagaagt
     3301 tgacttcgct ggttggctgt gctctaccat cggtctgaac cagccgtcta ccccgaccca
     3361 cgctgctggt gtggcagccg cagctgcgca tcatcaccac catcactaag gcgcgccgga
     3421 tccgaattcg agctccgtcg acaagctagc ttgatccggc tgctaacaaa gcccgaaagg
     3481 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3541 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatct ggcgtaatag
     3601 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     3661 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3721 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3781 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3841 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3901 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3961 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttagg
     4021 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4081 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4141 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4201 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4261 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4321 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4381 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4441 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4501 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4561 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4621 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4681 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4741 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4801 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4861 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4921 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4981 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5041 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5101 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5161 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5221 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5281 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5341 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5401 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5461 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5521 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5581 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5641 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5701 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5761 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5821 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5881 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5941 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     6001 c
//
