LOCUS       sequence-450701-        4044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Methanomethylophilus alvus pyrrolysine tRNA synthetase 
            engineered for 3-nitroY and 3-haloY under GlnS promoter. Used as a 
            control during Ma Pyl tRNA-synthetase validation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4044)
  TITLE     Engineering of Aminoacyl-tRNA synthetase/tRNA pairs for efficient 
            genetic encoding of non-canonical amino acids into proteins.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4044)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAJE-Ma pylRS nitroY/haloY-F5
FEATURES             Location/Qualifiers
     source          1..4044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      251..260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1608..1625)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1608..1624)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1617..1639)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1969..2013
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2051..2069)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2065..2112
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2283..3074)
                     /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(3075..3166)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3281..3868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3769..3788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgggagtt gtcagcctgt cccgcttata agatcatacg ccgttatacg ttgtttacgc
       61 tttgaggaat cccatatgac agtgaaatac acagatgccc agatccagcg cctgcgggag
      121 tatggcaacg gcacctatga gcagaaggtg tttgaagatc tggcctctag agatgcagcc
      181 ttctccaagg agatgtccgt ggcttccaca gacaacgaga aaaagatcaa gggcatgatt
      241 gccaacccca gccgccatgg gctgacccag ctgatgaatg acatcgccga cgccctggtg
      301 gccgagggct tcatcgaggt caggaccccc atcttcattt ctaaggacgc gctggctcgc
      361 atgaccatca ccgaggacaa gcccctgttc aagcaggtgt tctggatcga tgagaagagg
      421 gctctgaggc ccatgctcgc ccccaacctt tactccgtga tgcgggacct gcgcgaccac
      481 acggacggcc ctgtgaaaat tttcgaaatg ggctcctgct ttaggaaaga aagccacagc
      541 ggaatgcacc tggaggagtt caccatgctg gctctgtcgg acatggggcc aagaggagat
      601 gccacagaag tgctgaagaa ctacatctca gtggtcatga aggctgctgg actgcccgac
      661 tatgatttgg tgcaggaaga gagcgatgtc tacaaagaaa ccattgatgt ggagatcaat
      721 ggccaggagg tgtgctctgc ttgtgtgggc ccccactacc tggacgccgc ccacgacgtg
      781 catgaacccc gtagtggagc gggctttggc ctggagaggc tgctgaccat aagagaaaag
      841 tacagcactg tgaagaaagg cggcgcctcc atctcctact tgaatggagc caagatcaac
      901 agcggctgaa gcctgcagtt tcaaacgcta aattgcctga tgcgctacgc ttatcaggcc
      961 tacatgatct ctgcaatata ttgagtttgc gtgcttttgt aggccggata aggcgttcac
     1021 gccgcatccg gcaagaaaca gcaaacaatc caaaacgccg cgttcggcgg tcgacacaga
     1081 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     1141 gcagggcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     1201 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     1261 gatagtcatg ccccgcgccc accggaagga gctaccggca gcggtgcgga ctgttgtaac
     1321 tcagaataag aaatgaggcc gctcatggcg ttctgttgcc cgtctcactg gtgaaaagaa
     1381 aaacaaccct ggcgccgctt ctttgagcga acgatcaaaa ataagtggcg ccccatcaaa
     1441 aaaatattct caacataaaa aactttgtgt aatacttgta acgctagatc tgggggacgg
     1501 tccggcgacc agcgggtctc taaaacctag catagcgggg ttcgacaccc cggtctctcg
     1561 ccaactagta tccttagcga aagctaagga ttttttttaa gcttggcact ggccgtcgtt
     1621 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     1681 ccccctttcg ccagacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     1741 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gcccgagatg
     1801 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctggcg gccgcataat
     1861 gcttaagtcg aacagaaagt aatcgtattg tacacggccg cataatcgaa attaaattgg
     1921 atatcggcag gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa agaaaccgct
     1981 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     2041 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2101 aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta
     2161 gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     2221 ccgacgaccg ggtcatcgtg gccggatctt gcggcccctc ggcttgaacg aattgttaga
     2281 cattatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa ttcttccaac
     2341 tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg tctagcttca
     2401 agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc
     2461 ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca
     2521 tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc
     2581 gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc
     2641 aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg
     2701 gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg
     2761 cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca
     2821 gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa
     2881 gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc aatatcactg
     2941 tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt
     3001 tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac ttcggcgatc
     3061 accgcttccc tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3121 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagc tagcgggtgg
     3181 tgcgtaacgg caaaagcacc gccggacatc agcgctagcg gagtgtatac ttttcgttcc
     3241 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3301 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctttg acggtggttt gtttgccgga
     3361 tcaagagcgg ctaactcttt ttccgaaggt aactggctga cgcagagcgc agataccaaa
     3421 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3481 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3541 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3601 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3661 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3721 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3781 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3841 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3901 ggccttttgc tggccttttt ctagatttca gtgcaattta tctcttcaaa tgtagcacct
     3961 gaagtcagcc ccatacgata taagttgtaa ttctcatgtt tgacagctta tcatcgatgc
     4021 gaccgagtga gctagctatg gatc
//
