LOCUS       pDule2-Mj_Acd_9        6988 bp DNA     circular SYN 26-JAN-2023
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    pDule2-Tet2.0
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6988)
  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 6988)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 26, 2023 from SnapGene 6.2.0
            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"
FEATURES             Location/Qualifiers
     source          1..6988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..213
                     /label=pDule Mj Transfer for
                     /note="color: black; sequence: 
                     CGCTGAGTTTACGCTTTGAGGAATCCCCCATGGACGAATTTGAAATGAT; added: 
                     2022-01-13"
     CDS             194..1114
                     /codon_start=1
                     /label=Mj Acd A9
                     /note="color: #ffcc00"
                     /translation="MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGH
                     YLQIKKMIDLQNAGFDIIIDLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVY
                     GSELELDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNSIHYTG
                     VDVAVGGMEQRKINMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEI
                     RAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKE
                     LHPMDLKNAVAEELIKILEPIRKRL"
     misc_feature    287..289
                     /label=ala32
                     /note="color: #a6acb3"
     misc_feature    386..388
                     /label=asp 65
                     /note="color: #a6acb3"
     misc_feature    401..403
                     /label=his 70
                     /note="color: #a6acb3"
     misc_feature    515..517
                     /label=leu 108
                     /note="color: #a6acb3"
     misc_feature    518..520
                     /label=glu 109
                     /note="color: #a6acb3"
     misc_feature    665..667
                     /label=ser 158
                     /note="color: #a6acb3"
     misc_feature    668..670
                     /label=ile 159
                     /note="color: #a6acb3"
     misc_feature    677..679
                     /label=thr 162
                     /note="color: #a6acb3"
     misc_feature    722..724
                     /label=Asn 177
                     /note="color: #a6acb3"
     primer_bind     complement(1091..1149)
                     /label=pDule_Mj_Transfer_rev
                     /note="color: black; sequence: 
                     GTTCGGGCCCAAGCTTCGAATTCCCATATGGTACCTTATAATCTCTTTCTAATTGGCTC
                     ; added: 2022-01-13"
     CDS             1146..1175
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             1191..1208
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     primer_bind     complement(1264..1281)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
                     /note="color: black; sequence: GATTTAATCTGTATCAGG"
     terminator      1434..1520
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
     primer_bind     complement(1727..1744)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     rep_origin      complement(1861..2406)
                     /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.
                     "
                     /note="color: #ffff00"
     primer_bind     complement(1941..1960)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
     promoter        2557..2648
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             2649..3440
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /note="color: #ccffcc"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     complement(3616..3635)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
                     /note="color: black; sequence: GGTGATGTCGGCGATATAGG"
     primer_bind     4761..4783
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
                     /note="color: black; sequence: CCCAGTCACGACGTTGTAAAACG"
     primer_bind     4775..4792
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /note="color: black; sequence: TGTAAAACGACGGCCAGT"
     primer_bind     4776..4792
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     tRNA            complement(4829..4915)
                     /label=Mj tRNA
                     /note="color: #228b22; direction: LEFT"
     misc_feature    4916..4970
                     /label=lpp promoter
                     /note="color: #a6acb3"
     primer_bind     5776..5798
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
                     /note="color: black; sequence: CCGGGAGCTGCATGTGTCAGAGG"
     CDS             complement(5954..6832)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /note="color: #993366"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     6498..6507
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /note="color: black; sequence: TGTAAAACGACGGCCAGT"
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 tctgctgcga taggttttga
      301 accaagtggt aaaatacatt tagggcatta tctccaaata aaaaagatga ttgatttaca
      361 aaatgctgga tttgatataa ttatagattt ggctgattta cacgcctatt taaaccagaa
      421 aggagagttg gatgagatta gaaaaatagg agattataac aaaaaagttt ttgaagcaat
      481 ggggttaaag gcaaaatatg tttatggaag tgaactggag cttgataagg attatacact
      541 gaatgtctat agattggctt taaaaactac cttaaaaaga gcaagaagga gtatggaact
      601 tatagcaaga gaggatgaaa atccaaaggt tgctgaagtt atctatccaa taatgcaggt
      661 taattctatt cattatactg gcgttgatgt tgcagttgga gggatggagc agagaaaaat
      721 aaacatgtta 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 ataaggtacc atatgggaat tcgaagcttg
     1141 ggcccgaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc
     1201 atcatcattg agtttaaacg gtctccagct tggctgtttt ggcggatgag agaagatttt
     1261 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     1321 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     1381 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     1441 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     1501 ctctcctgag taggacaaat ccgccgggag ctgtccctcc tgttcagcta ctgacggggt
     1561 ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag cggagtgtat actggcttac
     1621 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg
     1681 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact
     1741 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     1801 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     1861 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     1921 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg
     1981 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     2041 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     2101 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2161 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     2221 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     2281 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     2341 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     2401 tctcaagaag atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc
     2461 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgtttg
     2521 acagcttatc atcgatgcga ccgagtgagc tagctatttg tttatttttc taaatacatt
     2581 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2641 ggaagagtat gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg
     2701 gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag
     2761 tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc
     2821 ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg
     2881 gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc
     2941 cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
     3001 ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag
     3061 caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc
     3121 ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg
     3181 actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag
     3241 taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg
     3301 cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
     3361 gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca
     3421 ccaaggtagt cggcaaataa tgtctaacaa ttcgttcaag ccgaggggcc gcaagatccg
     3481 gccacgatga cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt
     3541 gctggtttac cgggatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     3601 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     3661 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     3721 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     3781 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgaccgatgc
     3841 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     3901 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     3961 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     4021 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     4081 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct
     4141 tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg
     4201 gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc
     4261 agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc attggaccgc
     4321 tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg
     4381 taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg
     4441 ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat
     4501 tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat
     4561 atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggct ccttgcatgc
     4621 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     4681 gcaggagtcg cataagggag agcgtctggc gaaaggggga tgtgctgcaa ggcgattaag
     4741 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct
     4801 taaaaaaaat ccttagcttt cgctaaggat ctgcagtggt ccggcgggcc ggatttgaac
     4861 cagcgccatg cggatttaga gtccgccgtt ctgccctgct gaactaccgc cgggaattca
     4921 gcgttacaag tattacacaa agttttttat gttgagaata tttttttgat ggggcgccac
     4981 ttatttttga tcgttcgctc aaagaagcgg cgccagggtt gtttttcttt tcaccagtga
     5041 gacgggcaac agaacgccat gagcggcctc atttcttatt ctgagttaca acagtccgca
     5101 ccgctgccgg tagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     5161 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgcccaac agtcccccgg
     5221 ccacggggcc tgccaccata cccacgccga aacaagcgcc ctgcaccatt atgttccgga
     5281 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     5341 gctaaccgtt tttatcatgc tctgggaggc agaataaatg atcatatcgt caattattac
     5401 ctccacgggg agagcctgag caaactggcc tcaggcattt gagaagcaca cggtcacact
     5461 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     5521 ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta
     5581 ttatcactta ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa
     5641 aattacgccc cgccctgcca ctcatcgcag ttgactgggt catggctgcg ccccgacacc
     5701 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5761 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5821 gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc
     5881 aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct
     5941 gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg
     6001 cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt
     6061 gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa
     6121 gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact
     6181 gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg
     6241 cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc
     6301 gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt
     6361 gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa
     6421 tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc
     6481 ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa
     6541 agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct
     6601 ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt
     6661 tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc
     6721 ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat
     6781 gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca
     6841 tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact
     6901 gcgtctttta ctggctcttc tcgctaacca aaccggtaac ccgggaccaa gtttactcat
     6961 atatacggac agcggtgcgg actgttgt
//
