LOCUS       sequence-393189-        5082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express the variant of Methanosarcina mazei PylRS specific for 
            pyrrolysine derivatives (Boc-Lys, Az-ZLys, AzAmZLys) and its cognate
            Pyl tRNA and allow UAG codon to be translated into these derivati.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5082)
  AUTHORS   Yamaguchi A, Matsuda T, Ohtake K, Yanagisawa T, Yokoyama S, Fujiwara
            Y, Watanabe T, Hohsaka T, Sakamoto K
  TITLE     Incorporation of a Doubly Functionalized Synthetic Amino Acid into 
            Proteins for Creating Chemical and Light-Induced Conjugates.
  JOURNAL   Bioconjug Chem. 2016 Jan 20;27(1):198-206. doi: 
            10.1021/acs.bioconjchem.5b00602. Epub 2015 Dec 16.
  PUBMED    26625213
REFERENCE   2  (bases 1 to 5082)
  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"; doi: 
            "10.1021/acs.bioconjchem.5b00602"; journalName: "Bioconjug Chem"; 
            date: "2016-01-20- 20"; volume: "27"; issue: "1"; pages: "198-206"
COMMENT     Sequence Label: pCDF-Mm2
FEATURES             Location/Qualifiers
     source          1..5082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(4..18,19..24,25..41,42..48)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    24..40
                     /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    56..72
                     /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             91..102
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      1493..1587
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1968..2783)
                     /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(1968..2783)
                     /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     2691..2710
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2784..2875)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2923..3661)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3871..4953)
                     /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     4915..4934
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4954..5031)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaatcataaa aaatttattt gctttgtgag cggataacaa ttataataga ttcaattgtg
       61 agcggataac aatttcacac agaattcatt aaagaggaga aattaaccat ggataaaaaa
      121 ccactaaaca ctctgatatc tgcaaccggg ctctggatgt ccaggaccgg aacaattcat
      181 aaaataaaac accacgaagt ctctcgaagc aaaatctata ttgaaatggc atgcggagac
      241 caccttgttg taaacaactc caggagcagc aggactgcaa gagcgctcaa gcaccacaaa
      301 tacaggaaga cctgcaaacg ctgcagggtt tcggatgagg atctcaataa gttcctcaca
      361 aaggcaaacg aagaccagac aagcgtaaaa gtcaaggtcg tttctgcccc taccagaacg
      421 aaaaaggcaa tgccaaaatc cgttgcgaga gccccgaaac ctcttgagaa tacagaagcg
      481 gcacaggctc aaccttctga gtctaaattt tcacctgcga taccggtttc cacccaagag
      541 tcagtttctg tcccggcatc tgtttcaaca tcaatatcaa gcatttctac aggagcaact
      601 gcatccgcac tggtaaaagg gaatacgaac cccattacat ccatgtctgc ccctgttcag
      661 gcaagtgccc ccgcacttac gaagagccag actgacaggc ttgaagtcct gttaaaccca
      721 aaagatgaga tttccctgaa ttccggcaag cctttcaggg agcttgagtc cgaattgctc
      781 tctcgcagaa aaaaagacct gcagcagatc tacgcggaag aaagggagaa ttatctgggg
      841 aaactcgagc gtgaaattac caggttcttt gtggacaggg gttttctgga aataaaatcc
      901 ccgatcctga tccctcttga gtatatcgaa aggatgggca ttgataatga taccgaactt
      961 tcaaaacaga tcttcagggt tgacaagaac ttctgcctga gacccatgct tgctccaaac
     1021 cttgccaact acctgcgcaa gcttgacagg gccctgcctg atccaataaa aatttttgaa
     1081 ataggcccat gctacagaaa agagtccgac ggcaaagaac acctcgaaga gtttaccatg
     1141 ctgaacttct gccagatggg atcgggatgc acacgggaaa atcttgaaag cataattaca
     1201 gacttcctga accacctggg aattgatttc aagatcgtag gcgattcctg catggtcttt
     1261 ggggataccc ttgatgtaat gcacggagac ctggaacttt cctctgcagt agtcggaccc
     1321 ataccgcttg accgggaatg gggtattgat aaaccctgga taggggcagg tttcgggctc
     1381 gaacgccttc tcaaggttaa acacgacttt aaaaatatca agagagctgc aaggtccgaa
     1441 tcttactata acgggatttc taccaacctg taagcttaat tagctgagct tggactcctg
     1501 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc
     1561 cgccgggcgt tttttattgg tgagaatgat atccacaatc gagcttcttt gagcgaacga
     1621 tcaaaaataa gtggcgcccc atcaaaaaaa tattgacaac ataaaaaact ttgtgttata
     1681 attgtaacgg aaacctgatc atgtagatcg aatggactct aaatccgttc agccgggtta
     1741 gattcccggg gtttccgcca atccttagcg aaagctaagg atttttttta ataaaccggt
     1801 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgacaagctg
     1861 acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1921 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     1981 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     2041 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     2101 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     2161 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     2221 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     2281 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     2341 cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     2401 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     2461 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     2521 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     2581 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2641 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2701 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2761 gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag catttatcag
     2821 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaatagct
     2881 agctcactcg gtcgctacgc tccgggcgtg agactgcggc gggcgctgcg gacacataca
     2941 aagttaccca cagattccgt ggataagcag gggactaaca tgtgaggcaa aacagcaggg
     3001 ccgcgccggt ggcgtttttc cataggctcc gccctcctgc cagagttcac ataaacagac
     3061 gcttttccgg tgcatctgtg ggagccgtga ggctcaacca tgaatctgac agtacgggcg
     3121 aaacccgaca ggacttaaag atccccaccg tttccggcgg gtcgctccct cttgcgctct
     3181 cctgttccga ccctgccgtt taccggatac ctgttccgcc tttctccctt acgggaagtg
     3241 tggcgctttc tcatagctca cacactggta tctcggctcg gtgtaggtcg ttcgctccaa
     3301 gctgggctgt aagcaagaac tccccgttca gcccgactgc tgcgccttat ccggtaactg
     3361 ttcacttgag tccaacccgg aaaagcacgg taaaacgcca ctggcagcag ccattggtaa
     3421 ctgggagttc gcagaggatt tgtttagcta aacacgcggt tgctcttgaa gtgtgcgcca
     3481 aagtccggct acactggaag gacagatttg gttgctgtgc tctgcgaaag ccagttacca
     3541 cggttaagca gttccccaac tgacttaacc ttcgatcaaa ccacctcccc aggtggtttt
     3601 ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3661 cttttctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     3721 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     3781 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3841 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3901 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3961 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4021 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4081 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4141 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4201 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4261 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4321 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4381 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4441 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4501 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4561 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4621 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4681 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4741 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4801 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4861 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4921 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4981 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5041 gacgctctcc cttatgcgac tcctgcatta ggaaatctcg ag
//
