LOCUS       sequence-446767-        3274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA synthetase/tRNA pair for the in vivo incorporation of 
            4-Cyano-L-Tryptophan (4-CN-Trp) 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   Qianzhu H, Abdelkader EH, Welegedara AP, Habel E, Paul N, Frkic RL, 
            Jackson CJ, Huber T, Otting G
  TITLE     Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 
            4-Cyanotryptophan Conserves Their Structure and Enables the 
            Detection of Ligand Binding Sites.
  JOURNAL   Angew Chem Int Ed Engl. 2024 Dec 4:e202421000. doi: 
            10.1002/anie.202421000.
  PUBMED    39632265
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: "Angew Chem 
            Int Ed Engl. 2024 Dec 4:e202421000. doi: 10.1002/anie.202421000."
COMMENT     Sequence Label: pRSF-G1(4CNW)RS
FEATURES             Location/Qualifiers
     source          1..3274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11..29
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             61..83
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(988..1006)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1002..1049
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      complement(1530..2279)
                     /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(1540..1557)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1780..1799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2295..3110)
                     /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(2295..3110)
                     /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     3018..3037
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3111..3202)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tggtacccaa taatacgact cactataggg agaccacaac ggtttccctc tagaaataat
       61 tttgtttaac tttaagaagg agatatacat atggtggtga aatttaccga tagccagatt
      121 cagcatctga tggaatatgg tgataatgat tggagcgaag ccgaatttga agatgcagca
      181 gcacgtgata aagaatttag cagccagttt agcaaactga aaagcgccaa tgataaaggc
      241 ctgaaagatg ttattgcaaa tccgcgtaat gatctgaccg atctggaaaa caaaattcgc
      301 gaaaaactgg cagcccgtgg ttttattgaa gttcataccc cgatttttgt gagcaaaagc
      361 gcactggcaa aaatgaccat taccgaagat catccgctgt tcaaacaggt gttttggatt
      421 gatgataaac gtgcactgcg tccgatgcat gcaatgaata tttttaaagt tatgcgtgaa
      481 ctgcgcgatc ataccaaagg tccggttaaa atctttgaaa ttggtagctg ctttcgcaaa
      541 gaaagcaaaa gcagtaccca tctggaagaa tttaccatgc tgggcctggc cgaaatgggt
      601 cctgatggtg atccgatgga acatctgaaa atgtatattg gcgatatcat ggatgccgtt
      661 ggtgttgaat ataccaccag tcgtgaagaa tcagatgttt atgttgaaac cctggacgtg
      721 gaaattaatg gcaccgaagt tgcaagcggt actgttggtc cgcataaact ggatccggca
      781 catgatgtgc atgaaccgtc ggcaggtatt ggttttggtc tggaacgtct gctgatgctg
      841 aaaaatggta aaagcaatgc acgcaaaacc ggcaaaagta ttacctatct gaatggctac
      901 aaactggatt aagaattcga gctcccgggt accagataga tccggctgct aacaaagccc
      961 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1021 cctctaaacg ggtcttgagg ggttttttgg tttgtgagct cccgtaattc cgcttcgcaa
     1081 catgtgagca ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc
     1141 tgaggccagt ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcagtgca
     1201 cggctaacta agcggcctgc tgactttctc gccgatcaaa aggcattttg ctattaaggg
     1261 attgacgagg gcgtatctgc gcagtaagat gcgccccgca ttggagggcg ctccggcgag
     1321 caaacgggtc tctaaaacct gtaagcgggg ttcgaccccc cggcctttcg ccaaattcga
     1381 aaagcctgct caacgagcag gcttttttgc atgctcgagc agctcagggt cgaatttgct
     1441 ttcgaatttc tgccattcat ccgcttatta tcacttattc aggcgtagca accaggcgtt
     1501 taagggcacc aataactgcc ttaaaaaaac ttccgcttcc tcgctcactg actcgctacg
     1561 ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa tacggttatc
     1621 cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag caccggaaga
     1681 agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1741 gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1801 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1861 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg tatctcagtt
     1921 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1981 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2041 cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt atgcacctgt
     2101 taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca cttaccttgg
     2161 ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc ggtggttttt
     2221 ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc tattccgttg
     2281 aactgtggta cgcgttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2341 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     2401 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     2461 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     2521 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     2581 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     2641 ttcattcgtg attgcgcctg agcgagacga aatacgcggt cgctgttaaa aggacaatta
     2701 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2761 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     2821 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2881 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2941 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     3001 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     3061 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt
     3121 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3181 tgtatttaga aaaataaaca aataggcatg cagcgcttga cttgacggga cggcgttgta
     3241 attctcatgt ttgacagctt atcatcgata agct
//
