LOCUS       62056_20305        3170 bp DNA     circular SYN 19-APR-2020
DEFINITION  Cloning vector pSyn_Mj_sfGFP_200(amber), complete sequence.
ACCESSION   MN882183
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3170)
  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 3170)
  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 3170)
  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..3170
                     /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)"
     CDS             1546..1635
                     /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      1686..1733
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      1763..1781
                     /note="promoter for bacteriophage T7 RNA polymerase; T7 
                     promoter"
                     /regulatory_class="promoter"
     protein_bind    1782..1806
                     /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    1818..1822
                     /label=5' proK leader
                     /note="5' proK leader"
     misc_feature    1823..1899
                     /label=Methanocaldococcus jannaschii tRNApAzF(CTA)
                     /note="Methanocaldococcus jannaschii tRNApAzF(CTA)"
     misc_feature    1900..1938
                     /label=3' proK trailer
                     /note="3' proK trailer"
     regulatory      1939..1986
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase; T7 terminator"
                     /regulatory_class="terminator"
     regulatory      2024..2130
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2151..2226
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2234..2269
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     regulatory      2282..2387
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2406..2477
                     /note="guide RNA scaffold for the CRISPR/Cas9 system; gRNA 
                     scaffold"
     regulatory      2485..2520
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     promoter        2554..2601
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             2620..2991
                     /label=BleoR
                     /note="antibiotic-binding protein"
     terminator      3006..3100
                     /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 ctgcccgata atcactagct ctctactcag
     1441 agtgttttat cgaaagaccc gaacgagaag cgtgatcaca tggtgctgct tgaatttgtt
     1501 accgcggcag gtattacaca cggcatggat gagttgtata agggatccgc ttggagccac
     1561 ccgcagttcg agaaaggtgg aggttccgga ggtggatcgg gaggttcggc gtggagccac
     1621 ccgcagttcg aaaaataaaa gcttaattag ctgagcttgg actccctgcc accgctgagc
     1681 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1741 gaggaactat atccggattg gttaatacga ctcactatag gggaattgtg agcggataac
     1801 aattcccctc tagaaaagca ttccggcggt agttcagcag ggcagaacgg cggactctaa
     1861 atccgcatgg caggggttca aatcccctcc gccggaccaa attcgaaaag cctgctcaac
     1921 gagcaggctt ttttgcatct agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1981 tttttgcctg aacgagcagg cttttttgca taagcttcct agtggcagcg gctaactaag
     2041 cggcctgctg actttctcgc cgatcaaaag gcattttgct attaagggat tgacgagggc
     2101 gtatctgcgc agtaagatgc gccccgcatt ggagacgcca tggcgtctcg gttttagagc
     2161 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     2221 cggtgctttt tttaattcga aaagcctgct caacgagcag gcttttttgg tcgacagtag
     2281 tggcagcggc taactaagcg gcctgctgac tttctcgccg atcaaaaggc attttgctat
     2341 taagggattg acgagggcgt atctgcgcag taagatgcgc cccgcatgag acggcatgcc
     2401 gtctctagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     2461 tggcaccgag tcggtgcttt ttttaattcg aaaagcctgc tcaacgagca ggcttttttg
     2521 gtcgacagtt cataggtgat tgcggatccc gtcgttgaca attaatcatc ggcatagtat
     2581 atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc
     2641 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
     2701 gggttctccc gcgacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
     2761 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
     2821 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
     2881 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
     2941 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgagagctc
     3001 gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag
     3061 aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagcacta gctagtaaca
     3121 acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat
//
