LOCUS       62056_20310        3113 bp DNA     circular SYN 19-APR-2020
DEFINITION  Cloning vector pSyn_Mj_sfGFP_200(dest), complete sequence.
ACCESSION   MN882186
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3113)
  AUTHORS   Fischer EC, Hashimoto K, Zhang Y, Feldman AW, Dien VT, Karadeema RJ,
            Adhikary R, Ledbetter MP, Krishnamurthy R, Romesberg FE.
  TITLE     New codons for efficient production of unnatural proteins in a 
            semi-synthetic organism
  JOURNAL   Nat. Chem. Biol. (2020) In press
REFERENCE   2  (bases 1 to 3113)
  AUTHORS   Fischer EC.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-DEC-2019) Department of Chemistry, The Scripps 
            Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 
            92037, USA
REFERENCE   3  (bases 1 to 3113)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. Chem. 
            Biol. (2020) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-DEC-2019) Department of Chemistry, The Scripps Research 
            Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA"
FEATURES             Location/Qualifiers
     source          1..3113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      48..593
                     /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."
     promoter        746..764
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    766..784
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             799..821
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    1413..1430
                     /label=Y200 Golden Gate destination site
                     /note="Y200 Golden Gate destination site"
     CDS             1523..1612
                     /label=Twin-Strep-tag
                     /note="two Strep-Tag(R)II moieties connected by a linker 
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
     terminator      1663..1710
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      1740..1758
                     /note="promoter for bacteriophage T7 RNA polymerase; T7 
                     promoter"
                     /regulatory_class="promoter"
     protein_bind    1759..1783
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    1795..1799
                     /label=5' proK leader
                     /note="5' proK leader"
     misc_feature    1800..1842
                     /label=Methanocaldococcus jannaschii tRNApAzF(dest)
                     /note="Methanocaldococcus jannaschii tRNApAzF(dest)"
     misc_feature    1810..1827
                     /label=tRNApAzF Golden Gate destination site
                     /note="tRNApAzF Golden Gate destination site"
     misc_feature    1843..1881
                     /label=3' proK trailer
                     /note="3' proK trailer"
     regulatory      1882..1929
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase; T7 terminator"
                     /regulatory_class="terminator"
     regulatory      1967..2073
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2094..2169
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2177..2212
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     regulatory      2225..2330
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2349..2420
                     /note="guide RNA scaffold for the CRISPR/Cas9 system; gRNA 
                     scaffold"
     regulatory      2428..2463
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     promoter        2497..2544
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             2563..2934
                     /label=BleoR
                     /note="antibiotic-binding protein"
     terminator      2949..3043
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 tgcactgaaa tctagtaata ttttatctga ttaataagat gatcttcttg agatcgtttt
       61 ggtctgcgcg taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg
      121 aaggttctct gagctaccaa ctctttgaac cgaggtaact ggcttggagg agcgcagtca
      181 ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct
      241 aaatcaatta ccagtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc
      301 aagacgatag ttaccggata aggcgcagcg gtcggactga acggggggtt cgtgcataca
      361 gtccagcttg gagcgaactg cctacccgga actgagtgtc aggcgtggaa tgagacaaac
      421 gcggccataa cagcggaatg acaccggtaa accgaaaggc aggaacagga gagcgcacga
      481 gggagccgcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccaccact
      541 gatttgagcg tcagatttcg tgatgcttgt caggggggcg gagcctatgg aaaaacggct
      601 ttgccgcggc cctctcactt ccctgttaag tatcttcctg gcatcttcca ggaaatctcc
      661 gccccgttcg taagccattt ccgctcgccg cagtcgaacg accgagcgta gcgagtcagt
      721 gagcgaggaa gcggaatata tcccttaata cgactcacta tagggtccct atcagtgata
      781 gagaggtcta gaaataattt tgtttaactt taagaaggag atatacatat gtcgaaaggc
      841 gaagaactgt ttacgggagt ggtgcctatc ctggtagagc tcgacggaga tgtaaacggt
      901 cacaaatttt cagtccgcgg ggaaggcgaa ggcgatgcga ccaacggtaa attaactttg
      961 aagtttattt gcaccaccgg caaattaccg gtgccttggc cgacgcttgt gacgaccctg
     1021 acttacgggg tgcagtgttt cagtcgctac ccagatcaca tgaaacgcca tgacttcttc
     1081 aaatctgcga tgccggaagg ctatgtgcag gaacgtacaa ttagctttaa agacgacggc
     1141 acgtataaaa cgcgggcaga ggttaaattt gagggagata ccctggtaaa ccgtattgaa
     1201 ctgaaaggca tcgattttaa agaagatggg aacatcttgg gccacaaact cgagtacaac
     1261 tttaactcac acaatgtata catcacggca gacaaacaaa agaatggaat caaagcaaat
     1321 ttcaagatcc gtcataacgt ggaggacggt tccgtgcagc ttgcagatca ctatcagcag
     1381 aatacgccga ttggcgatgg cccggtgctg ctgagaccgg taccggtctc aagacccgaa
     1441 cgagaagcgt gatcacatgg tgctgcttga atttgttacc gcggcaggta ttacacacgg
     1501 catggatgag ttgtataagg gatccgcttg gagccacccg cagttcgaga aaggtggagg
     1561 ttccggaggt ggatcgggag gttcggcgtg gagccacccg cagttcgaaa aataaaagct
     1621 taattagctg agcttggact ccctgccacc gctgagcaat aactagcata accccttggg
     1681 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggtt
     1741 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaaagcattc
     1801 cggcggtagg agaccggtac cggtctcccc tccgccggac caaattcgaa aagcctgctc
     1861 aacgagcagg cttttttgca tctagcataa ccccttgggg cctctaaacg ggtcttgagg
     1921 ggttttttgc ctgaacgagc aggctttttt gcataagctt cctagtggca gcggctaact
     1981 aagcggcctg ctgactttct cgccgatcaa aaggcatttt gctattaagg gattgacgag
     2041 ggcgtatctg cgcagtaaga tgcgccccgc attggagacg ccatggcgtc tcggttttag
     2101 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2161 agtcggtgct ttttttaatt cgaaaagcct gctcaacgag caggcttttt tggtcgacag
     2221 tagtggcagc ggctaactaa gcggcctgct gactttctcg ccgatcaaaa ggcattttgc
     2281 tattaaggga ttgacgaggg cgtatctgcg cagtaagatg cgccccgcat gagacggcat
     2341 gccgtctcta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     2401 aagtggcacc gagtcggtgc tttttttaat tcgaaaagcc tgctcaacga gcaggctttt
     2461 ttggtcgaca gttcataggt gattgcggat cccgtcgttg acaattaatc atcggcatag
     2521 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
     2581 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     2641 ctcgggttct cccgcgactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     2701 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     2761 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     2821 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     2881 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgagag
     2941 ctcgcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt
     3001 cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagca ctagctagta
     3061 acaacttata tcgtatgggg ctgacttcag gtgctacatt tgaagagata aat
//
