LOCUS       sequence-169854-        7003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for incorporating the non-canonical amino acid Tetrazine 2.0
            with the Mj Tet2.0 synthetase and cognate amber suppressing tRNA in 
            Ecoli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7003)
  AUTHORS   Blizzard RJ, Backus DR, Brown W, Bazewicz CG, Li Y, Mehl RA
  TITLE     Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded 
            Tetrazine Amino Acid.
  JOURNAL   J Am Chem Soc. 2015 Aug 19;137(32):10044-7. doi: 
            10.1021/jacs.5b03275. Epub 2015 Aug 10.
  PUBMED    26237426
REFERENCE   2  (bases 1 to 7003)
  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/jacs.5b03275"; journalName: "J Am Chem Soc"; date: 
            "2015-08-19- 19"; volume: "137"; issue: "32"; pages: "10044-7"
COMMENT     Sequence Label: pDule2-Tet2.0
FEATURES             Location/Qualifiers
     source          1..7003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1161..1190
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1206..1223
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1279..1296)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1449..1535
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1742..1759)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1876..2421)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1956..1975)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2572..2663
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2664..3455
                     /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(3631..3650)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4776..4798
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4791..4807
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5791..5813
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5969..6847)
                     /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 ttatacagtt ggctgattta cacgcctatt taaaccagaa
      421 aggagagttg gatgagatta gaaaaatagg agattataac aaaaaagttt ttgaagcaat
      481 ggggttaaag gcaaaatatg tttatggaag tgaatctgat cttgataagg attatacact
      541 gaatgtctat agattggctt taaaaactac cttaaaaaga gcaagaagga gtatggaact
      601 tatagcaaga gaggatgaaa atccaaaggt tgctgaagtt atctatccaa taatgcaggt
      661 taattctatt 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 ataactgcag tttcaaacgg gtaccatatg
     1141 ggaattcgaa gcttgggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg
     1201 tcgaccatca tcatcatcat cattgagttt aaacggtctc cagcttggct gttttggcgg
     1261 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     1321 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     1381 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     1441 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1501 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagctgtc cctcctgttc
     1561 agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag
     1621 tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca
     1681 ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt
     1741 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg
     1801 aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc
     1861 cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg
     1921 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1981 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt
     2041 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca
     2101 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact
     2161 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta
     2221 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca
     2281 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag
     2341 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg
     2401 cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt
     2461 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     2521 taattctcat gtttgacagc ttatcatcga tgcgaccgag tgagctagct atttgtttat
     2581 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2641 aataatattg aaaaaggaag agtatgaggg aagcggtgat cgccgaagta tcgactcaac
     2701 tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt
     2761 tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta
     2821 cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa
     2881 cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc
     2941 acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc
     3001 agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta
     3061 tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac
     3121 tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat
     3181 ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca
     3241 tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa
     3301 tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg
     3361 gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg
     3421 tgaaaggcga gatcaccaag gtagtcggca aataatgtct aacaattcgt tcaagccgag
     3481 gggccgcaag atccggccac gatgacccgg tcgtcggttc agggcagggt cgttaaatag
     3541 ccgcttatgt ctattgctgg tttaccggga tcctctacgc cggacgcatc gtggccggca
     3601 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     3661 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     3721 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     3781 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     3841 agcgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     3901 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     3961 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     4021 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     4081 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg
     4141 cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga
     4201 ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg
     4261 tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt
     4321 cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg
     4381 ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg
     4441 gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt
     4501 caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa
     4561 cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc
     4621 gggctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     4681 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt ctggcgaaag ggggatgtgc
     4741 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     4801 ggccagtgcc aagcttaaaa aaaatcctta gctttcgcta aggatctgca gtggtccggc
     4861 gggccggatt tgaaccagcg ccatgcggat ttagagtccg ccgttctgcc ctgctgaact
     4921 accgccggga attcagcgtt acaagtatta cacaaagttt tttatgttga gaatattttt
     4981 ttgatggggc gccacttatt tttgatcgtt cgctcaaaga agcggcgcca gggttgtttt
     5041 tcttttcacc agtgagacgg gcaacagaac gccatgagcg gcctcatttc ttattctgag
     5101 ttacaacagt ccgcaccgct gccggtagct ccttccggtg ggcgcggggc atgactatcg
     5161 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     5221 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgccctgca
     5281 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     5341 ctgtattaac gaagcgctaa ccgtttttat catgctctgg gaggcagaat aaatgatcat
     5401 atcgtcaatt attacctcca cggggagagc ctgagcaaac tggcctcagg catttgagaa
     5461 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     5521 ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg
     5581 ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta agggcaccaa
     5641 taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagttgac tgggtcatgg
     5701 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5761 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5821 cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg
     5881 cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca
     5941 agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt
     6001 tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc
     6061 gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc
     6121 gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga
     6181 cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat
     6241 gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac
     6301 aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc
     6361 gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc
     6421 cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat
     6481 ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt
     6541 aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac
     6601 cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt
     6661 ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt
     6721 tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta
     6781 aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt
     6841 cagccatact tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca
     6901 gacattgccg tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaacccggg
     6961 accaagttta ctcatatata cggacagcgg tgcggactgt tgt
//
