LOCUS       sequence-400630-        4928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid encodes an orthogonal aminoacyl-tRNA synthetase/tRNA-CUA
            pair for amber codon suppression with N-epsilon-Boc-lysine..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4928)
  AUTHORS   Fricke R, Swenson CV, Roe LT, Hamlish NX, Shah B, Zhang Z, Ficaretta
            E, Ad O, Smaga S, Gee CL, Chatterjee A, Schepartz A
  TITLE     Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase 
            enzymes to include non-alpha-amino acids in vitro and in vivo.
  JOURNAL   Nat Chem. 2023 Jun 1. doi: 10.1038/s41557-023-01224-y.
  PUBMED    37264106
REFERENCE   2  (bases 1 to 4928)
  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: "Nat Chem. 
            2023 Jun 1. doi: 10.1038/s41557-023-01224-y."
COMMENT     Sequence Label: pMega-MaPylRS
FEATURES             Location/Qualifiers
     source          1..4928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(26..103)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(151..156,157..172,173..179)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    187..203
                     /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)."
     primer_bind     complement(1115..1132)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     protein_bind    1604..1620
                     /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)."
     regulatory      1800..1809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1992..2783)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     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(join(3871..4928,1..25))
                     /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     join(4915..4928,1..6)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
       61 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
      121 cccttatgcg actcctgcat tagggagctg ttgacaatta atcatcggct cgtataatgt
      181 gtggaattgt gagcggataa caatttcaca aaggaggtgc ggccgcatga cagtcaaata
      241 taccgacgcc cagattcagc gtttgcgcga gtacggcaac ggtacgtatg agcaaaaggt
      301 ttttgaagac cttgctagtc gcgacgcggc gttcagcaag gaaatgagtg ttgcctccac
      361 agataatgaa aagaaaatca aaggcatgat cgccaacccc tctcgtcacg gcttgaccca
      421 gctgatgaac gatattgcag atgcgttggt agctgagggg ttcattgagg tgcgtactcc
      481 tattttcatt agtaaggacg cccttgcacg catgactatt acggaggata aacctttgtt
      541 caaacaggtc ttctggatcg atgagaagcg tgcactgcgc cccatgctgg cacccaactt
      601 atatagtgtc atgcgcgatt tacgcgatca cactgacggt ccggtgaaaa tttttgagat
      661 gggctcatgc ttccgcaaag agtcgcattc tggtatgcac ctggaagagt ttaccatgtt
      721 aaacctggta gacatggggc cacgcgggga cgctacggag gtcctgaaga actacattag
      781 cgtcgtgatg aaagcagccg ggttacccga ttacgatctt gttcaggaag agagtgacgt
      841 ttacaaagaa acaatcgatg ttgaaatcaa cggtcaagaa gtttgttcag cggccgtggg
      901 gccgcattat ttagacgctg cccacgatgt acatgagccg tggagtggtg caggcttcgg
      961 tcttgagcgc ttgctgacca ttcgtgagaa atatagcact gtcaaaaaag gtggcgcctc
     1021 catctcttat cttaatggag ctaaaatcaa ctaagcggcc gcgtttaaac ggtctccagc
     1081 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     1141 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     1201 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     1261 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1321 ttgtttgtga gctcccggtc atcaatcatc cccataatcc ttgttagcct gcaggtaatt
     1381 ccgcttcgca acatgtgagc accggtttat tgactaccgg aagcagtgtg accgtgtgct
     1441 tctcaaatgc ctgaggccag tttgctcagg ctctccccgt ggaggtaata attgacgata
     1501 tgatcagtgc acggctaact aagcggcctg ctgactttct cgccgatcaa aaggcatttt
     1561 gctattaagg gattgacgag ggcgtatctg cgcagtaaga taattgtgag cggataacaa
     1621 ttagcagaca agatgggtcc cttatcatgg caaccatctg aacgggggac ggtccggcga
     1681 ccagcgggtc tctaaaacct agccagcggg gttcgacgcc ccggtctctc gccaaattcg
     1741 aaaagcctgc tcaacgagca ggcttttttg catgctcgag cagctcaggg tcgaatttgc
     1801 catggcggcc accaggtacc accggcgcct caggcatttg agaagcacac ggtcacactg
     1861 cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     1921 tgccctgaac cgacgaccgg gtcatcgtgg ccggatcttg cggcccctcg gcttgaacga
     1981 attgttagac attatttgcc gactaccttg gtgatctcgc ctttcacgta gtggacaaat
     2041 tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag ataagcctgt
     2101 ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc ccagtcggca
     2161 gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg ggacaacgta
     2221 agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag cgttaaggtt
     2281 tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc ctccgccgct
     2341 ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc cagatcaatg
     2401 tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat tgcgctgcca ttctccaaat
     2461 tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg
     2521 acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg
     2581 ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac cagcaaatca
     2641 atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg tacggccagc
     2701 aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg agtcgatact
     2761 tcggcgatca ccgcttccct 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 ctctgcga
//
