LOCUS       sequence-424910-        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the 2nd-generation ""E7"" Tet2 Methanocaldococcus 
            jannaschii (Mj) aminoacyl tRNA synthetase/tRNA pair for encoding 
            Tet2 noncanonical amino acids into TAG sites of proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  AUTHORS   Eddins AJ, Bednar RM, Jana S, Pung AH, Mbengi L, Meyer K, Perona JJ,
            Cooley RB, Karplus PA, Mehl RA
  TITLE     Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids 
            for Quantitative Protein Ligations.
  JOURNAL   Bioconjug Chem. 2023 Dec 20;34(12):2243-2254. doi: 
            10.1021/acs.bioconjchem.3c00380. Epub 2023 Dec 4.
  PUBMED    38047550
REFERENCE   2  (bases 1 to 6849)
  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.3c00380"; journalName: "Bioconjug Chem"; 
            date: "2023-12-20- 20"; volume: "34"; issue: "12"; pages: 
            "2243-2254"
COMMENT     Sequence Label: pAJE3-E7
FEATURES             Location/Qualifiers
     source          1..6849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1143..1172
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1188..1205
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1261..1278)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1431..1517
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1660..2247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2148..2167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2418..2509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2510..3301
                     /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"
     primer_bind     complement(3477..3496)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4622..4644
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4637..4653
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5637..5659
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5815..6693)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aactcagaat aagaaatgag gccgctcatg gcgttctgtt gcccgtctca ccggtgaaaa
       61 gaaaaacaac cctggcgccg cttctttgag cgaacgatca aaaataagtg gcgccccatc
      121 aaaaaaatat tctcaacata aaaaactttg tgtaatactt gtaacgctga gtttacgctt
      181 tgaggaatcc cccatggacg aatttgaaat gataaagaga aacacatctg aaattatcag
      241 cgaggaagag ttaagagagg ttttaaaaaa agatgaaaaa tctgctggga taggttttga
      301 accaagtggt aaaatacatt tagggcatta tctccaaata aaaaagatga ttgatttaca
      361 aaatgctgga tttgatataa ttatagcttt ggctgattta atggcctatt taaaccagaa
      421 aggagagttg gatgagatta gaaaaatagg agattataac aaaaaagttt ttgaagcaat
      481 ggggttaaag gcaaaatatg tttatggaag tgaatctgag cttgataagg attatacact
      541 gaatgtctat agattggctt taaaaactac cttaaaaaga gcaagaagga gtatggaact
      601 tatagcaaga gaggatgaaa atccaaaggt tgctgaagtt atctatccaa taatgcaggt
      661 taatggtatt cattataatg gcgttgatgt tgcagttgga gggatggagc agagaaaaat
      721 acacatgtta gcaagggagc ttttaccaaa aaaggttgtt tgtattcaca accctgtctt
      781 aacgggtttg gatggagaag gaaagatgag ttcttcaaaa gggaatttta tagctgttga
      841 tgactctcca gaagagatta gggctaagat aaagaaagca tactgcccag ctggagttgt
      901 tgaaggaaat ccaataatgg agatagctaa atacttcctt gaatatcctt taaccataaa
      961 aaggccagaa aaatttggtg gagatttgac agttaatagc tatgaggagt tagagagttt
     1021 atttaaaaat aaggaattgc atccaatgga tttaaaaaat gctgtagctg aagaacttat
     1081 aaagatttta gagccaatta gaaagagatt ataactcata tgggaattcg aagcttgggc
     1141 ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc
     1201 atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga agattttcag
     1261 cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg
     1321 cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc
     1381 cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac
     1441 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1501 tcctgagtag gacaaatccg ccgggagctg tccctcctgt tcagctactg acggggtggt
     1561 gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact tttcgttcca
     1621 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1681 cgtaatctgc tgcttgcaaa caaaaaaacc accgctttga cggtggtttg tttgccggat
     1741 caagagcggc taactctttt tccgaaggta actggctgac gcagagcgca gataccaaat
     1801 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1861 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1921 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1981 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2041 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2101 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2161 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2221 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2281 gccttttgct ggcctttttc tagatttcag tgcaatttat ctcttcaaat gtagcacctg
     2341 aagtcagccc catacgatat aagttgtaat tctcatgttt gacagcttat catcgatgcg
     2401 accgagtgag ctagctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2461 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagggaagc
     2521 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     2581 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     2641 acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     2701 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     2761 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     2821 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     2881 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     2941 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     3001 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     3061 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     3121 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     3181 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     3241 tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata
     3301 atgtctaaca attcgttcaa gccgaggggc cgcaagatcc ggccacgatg acccggtcgt
     3361 cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta ccgggatcct
     3421 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
     3481 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
     3541 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
     3601 gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt
     3661 cctaatgcag gagtcgcata agggagagcg tcgaccgatg cccttgagag ccttcaaccc
     3721 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     3781 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     3841 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     3901 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     3961 cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg
     4021 aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc
     4081 gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc
     4141 gctcgcggct cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta
     4201 tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg ccctatacct
     4261 tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga
     4321 agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg
     4381 cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc
     4441 tccagcagcc gcacgcggcg catctcgggc tccttgcatg caccattcct tgcggcggcg
     4501 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     4561 gagcgtctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     4621 tcccagtcac gacgttgtaa aacgacggcc agtgccaagc ttaaaaaaaa tccttagctt
     4681 tcgctaagga tctgcagtgg tccggcgggc cggatttgaa ccagcgccat gcggatttag
     4741 agtccgccgt tctgccctgc tgaactaccg ccgggaattc agcgttacaa gtattacaca
     4801 aagtttttta tgttgagaat atttttttga tggggcgcca cttatttttg atcgttcgct
     4861 caaagaagcg gcgccagggt tgtttttctt ttcaccagtg agacgggcaa cagaacgcca
     4921 tgagcggcct catttcttat tctgagttac aacagtccgc accgctgccg gtagctcctt
     4981 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     5041 actcgtagga caggtgccgg cagcgcccaa cagtcccccg gccacggggc ctgccaccat
     5101 acccacgccg aaacaagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     5161 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctaaccgt ttttatcatg
     5221 ctctgggagg cagaataaat gatcatatcg tcaattatta cctccacggg gagagcctga
     5281 gcaaactggc ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa
     5341 ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga accgacgacc
     5401 gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt
     5461 agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc
     5521 actcatcgca gttgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5581 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5641 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa
     5701 ttcgcgcgcg aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat
     5761 tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga
     5821 caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct
     5881 ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt
     5941 gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat
     6001 acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga
     6061 cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc
     6121 tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg
     6181 gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc
     6241 cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc
     6301 gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga
     6361 cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata
     6421 ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa
     6481 aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg
     6541 aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag
     6601 ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc
     6661 cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga
     6721 agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt
     6781 ctcgctaacc aaaccggtaa cccgggacca agtttactca tatatacgga cagcggtgcg
     6841 gactgttgt
//
