LOCUS       sequence-169865-        6333 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 6333)
  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 6333)
  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: pDule-Tet2.0
FEATURES             Location/Qualifiers
     source          1..6333
                     /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        join(2533..2538,2539..2555,2556..2561)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2609..3799
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(2961..2980)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4106..4128
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4121..4137
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5121..5143
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5299..6177)
                     /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 taagctttaa tgcggtagtt tatcacagtt
     2581 aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct
     2641 cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct
     2701 cttgcgggat atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct
     2761 atatgcgttg atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg
     2821 ccgccgccca gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc
     2881 gaccacaccc gtcctgtgga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     2941 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     3001 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     3061 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     3121 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgacc
     3181 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     3241 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     3301 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     3361 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     3421 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta
     3481 cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc
     3541 ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga
     3601 ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcattgg
     3661 accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg
     3721 gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag
     3781 ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca
     3841 agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag
     3901 aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggctccttg
     3961 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     4021 ctaatgcagg agtcgcataa gggagagcgt ctggcgaaag ggggatgtgc tgcaaggcga
     4081 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc
     4141 aagcttaaaa aaaatcctta gctttcgcta aggatctgca gtggtccggc gggccggatt
     4201 tgaaccagcg ccatgcggat ttagagtccg ccgttctgcc ctgctgaact accgccggga
     4261 attcagcgtt acaagtatta cacaaagttt tttatgttga gaatattttt ttgatggggc
     4321 gccacttatt tttgatcgtt cgctcaaaga agcggcgcca gggttgtttt tcttttcacc
     4381 agtgagacgg gcaacagaac gccatgagcg gcctcatttc ttattctgag ttacaacagt
     4441 ccgcaccgct gccggtagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     4501 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc ccaacagtcc
     4561 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgccctgca ccattatgtt
     4621 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     4681 gaagcgctaa ccgtttttat catgctctgg gaggcagaat aaatgatcat atcgtcaatt
     4741 attacctcca cggggagagc ctgagcaaac tggcctcagg catttgagaa gcacacggtc
     4801 acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa
     4861 cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc
     4921 gcttattatc acttattcag gcgtagcaac caggcgttta agggcaccaa taactgcctt
     4981 aaaaaaatta cgccccgccc tgccactcat cgcagttgac tgggtcatgg ctgcgccccg
     5041 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5101 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5161 gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg cataatgtgc
     5221 ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat
     5281 tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt tttcttcaca
     5341 accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata
     5401 gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct
     5461 caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc
     5521 taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac
     5581 gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg
     5641 tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa
     5701 caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc
     5761 gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa
     5821 gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg
     5881 acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg
     5941 aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg
     6001 atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag
     6061 cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac
     6121 cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact
     6181 tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg
     6241 tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaacccggg accaagttta
     6301 ctcatatata cggacagcgg tgcggactgt tgt
//
