LOCUS Exported 6333 bp ds-DNA circular SYN 12-MAY-2021 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 pDule-Tet2.0 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6333 /organism="synthetic DNA construct" /mol_type="other DNA" 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 2533..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 4120..4137 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" 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 //