LOCUS       sequence-393448-        3274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA synthetase/tRNA pair for the in vivo incorporation of 
            N6-(((trimethylsilyl)methyl)carbamoyl)-L-lysine (TMSNK), into 
            proteins in E. coli in response to the amber (TAG) codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3274)
  AUTHORS   Ekanayake KB, Mahawaththa MC, Qianzhu H, Abdelkader EH, George J, 
            Ullrich S, Murphy RB, Fry SE, Johansen-Leete J, Payne RJ, Nitsche C,
            Huber T, Otting G
  TITLE     Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic 
            Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino 
            Acids.
  JOURNAL   J Med Chem. 2023 Mar 15. doi: 10.1021/acs.jmedchem.3c00222.
  PUBMED    36920850
REFERENCE   2  (bases 1 to 3274)
  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: "J Med Chem.
            2023 Mar 15. doi: 10.1021/acs.jmedchem.3c00222."
COMMENT     Sequence Label: pRSF-G1TMSNKRS
FEATURES             Location/Qualifiers
     source          1..3274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(122..871)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(132..149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(372..391)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(887..1702)
                     /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(887..1702)
                     /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     1610..1629
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1703..1794)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1877..1896
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1877..1895
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1927..1949
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(2854..2872)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2868..2915
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gcatgctcga gcagctcagg gtcgaatttg ctttcgaatt tctgccattc atccgcttat
       61 tatcacttat tcaggcgtag caaccaggcg tttaagggca ccaataactg ccttaaaaaa
      121 acttccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg
      181 tcagctcact caaaagcggt aatacggtta tccacagaat caggggataa agccggaaag
      241 aacatgtgag caaaaagcaa agcaccggaa gaagccaacg ccgcaggcgt ttttccatag
      301 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc
      361 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      421 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      481 ttctcatagc tcacgctgtt ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      541 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      601 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccattg gtaactgatt
      661 tagaggactt tgtcttgaag ttatgcacct gttaaggcta aactgaaaga acagattttg
      721 gtgagtgcgg tcctccaacc cacttacctt ggttcaaaga gttggtagct cagcgaacct
      781 tgagaaaacc accgttggta gcggtggttt ttctttattt atgagatgat gaatcaatcg
      841 gtctatcaag tcaacgaaca gctattccgt tgaactgtgg tacgcgttag aaaaactcat
      901 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
      961 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1021 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1081 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1141 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1201 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1261 gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1321 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1381 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     1441 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     1501 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     1561 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     1621 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     1681 tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg
     1741 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggca
     1801 tgcagcgctt gacttgacgg gacggcgttg taattctcat gtttgacagc ttatcatcga
     1861 taagcttggt acccaataat acgactcact atagggagac cacaacggtt tccctctaga
     1921 aataattttg tttaacttta agaaggagat atacatatgg tggtgaaatt taccgatagc
     1981 cagattcagc atctgatgga atatggtgat aatgattgga gcgaagccga atttgaagat
     2041 gcagcagcac gtgataaaga atttagcagc cagtttagca aactgaaaag cgccaatgat
     2101 aaaggcctga aagatgttat tgcaaatccg cgtaatgatc tgaccgatct ggaaaacaaa
     2161 attcgcgaaa aactggcagc ccgtggtttt attgaagttc ataccccgat ttttgtgagc
     2221 aaaagcgcac tggcaaaaat gaccattacc gaagatcatc cgctgttcaa acaggtgttt
     2281 tggattgatg ataaacgtgc actgcgtccg atgcatgcaa tgaatctgta taaagttatg
     2341 cgtgaactgc gcgatcatac caaaggtccg gttaaaatct ttgaaattgg tagctgcttt
     2401 cgcaaagaaa gcaaaagcag tacccatctg gaagaattta ccatgctgaa cctgggcgaa
     2461 atgggtcctg atggtgatcc gatggaacat ctgaaaatgt atattggcga tatcatggat
     2521 gccgttggtg ttgaatatac caccagtcgt gaagaatcag atgtttgggt tgaaaccctg
     2581 gacgtggaaa ttaatggcac cgaagttgca agcggtgctg ttggtccgca taaactggat
     2641 ccggcacatg atgtgcatga accgtgggca ggtattggtt ttggtctgga acgtctgctg
     2701 atgctgaaaa atggtaaaag caatgcacgc aaaaccggca aaagtattac ctatctgaat
     2761 ggctacaaac tggattaaga attcgagctc ccgggtacca gatagatccg gctgctaaca
     2821 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2881 ttggggcctc taaacgggtc ttgaggggtt ttttggtttg tgagctcccg taattccgct
     2941 tcgcaacatg tgagcaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca
     3001 aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc
     3061 agtgcacggc taactaagcg gcctgctgac tttctcgccg atcaaaaggc attttgctat
     3121 taagggattg acgagggcgt atctgcgcag taagatgcgc cccgcattgg agggcgctcc
     3181 ggcgagcaaa cgggtctcta aaacctgtaa gcggggttcg accccccggc ctttcgccaa
     3241 attcgaaaag cctgctcaac gagcaggctt tttt
//
