LOCUS       62056_20325        3435 bp DNA     circular SYN 19-APR-2020
DEFINITION  Cloning vector pSyn_Mz_Mj_sfGFP_190_200(amber-ochre), complete 
            sequence.
ACCESSION   MN882184
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3435)
  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 3435)
  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 3435)
  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..3435
                     /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..1894
                     /note="tRNA-Pyl; Methanosarcina mazei tRNAPyl(TTA)"
     misc_feature    1895..1933
                     /label=3' proK trailer
                     /note="3' proK trailer"
     regulatory      1934..1981
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase; T7 terminator"
                     /regulatory_class="terminator"
     regulatory      2028..2046
                     /note="promoter for bacteriophage T7 RNA polymerase; T7 
                     promoter"
                     /regulatory_class="promoter"
     protein_bind    2047..2071
                     /label=lacI binding site
                     /bound_moiety="lacI"
                     /note="lac repressor binds to the lac operator to inhibit 
                     transcription in E. coli; lac operator"
     misc_feature    2083..2087
                     /label=5' proK leader
                     /note="5' proK leader"
     misc_feature    2088..2164
                     /label=Methanocaldococcus jannaschii tRNApAzF(CTA)
                     /note="Methanocaldococcus jannaschii tRNApAzF(CTA)"
     misc_feature    2165..2203
                     /label=3' proK trailer
                     /note="3' proK trailer"
     regulatory      2204..2251
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase; T7 terminator"
                     /regulatory_class="terminator"
     regulatory      2289..2395
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2416..2491
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2499..2534
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     regulatory      2547..2652
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2671..2742
                     /note="guide RNA scaffold for the CRISPR/Cas9 system; gRNA 
                     scaffold"
     regulatory      2750..2785
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     promoter        2819..2866
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             2885..3256
                     /label=BleoR
                     /note="antibiotic-binding protein"
     terminator      3271..3365
                     /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 ttggctaagg 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 ttggaaacct gatcatgtag atcgaacgga ctttaaatcc
     1861 gttcagccgg gttagattcc cggggtttcc gccaaattcg aaaagcctgc tcaacgagca
     1921 ggcttttttg catctagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1981 gcctgaacga gcaggctgct gaaaggagga actatatccg gattggttaa tacgactcac
     2041 tataggggaa ttgtgagcgg ataacaattc ccctctagaa aagcattccg gcggtagttc
     2101 agcagggcag aacggcggac tctaaatccg catggcaggg gttcaaatcc cctccgccgg
     2161 accaaattcg aaaagcctgc tcaacgagca ggcttttttg catctagcat aaccccttgg
     2221 ggcctctaaa cgggtcttga ggggtttttt gcctgaacga gcaggctttt ttgcataagc
     2281 ttcctagtgg cagcggctaa ctaagcggcc tgctgacttt ctcgccgatc aaaaggcatt
     2341 ttgctattaa gggattgacg agggcgtatc tgcgcagtaa gatgcgcccc gcattggaga
     2401 cgccatggcg tctcggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     2461 tcaacttgaa aaagtggcac cgagtcggtg ctttttttaa ttcgaaaagc ctgctcaacg
     2521 agcaggcttt tttggtcgac agtagtggca gcggctaact aagcggcctg ctgactttct
     2581 cgccgatcaa aaggcatttt gctattaagg gattgacgag ggcgtatctg cgcagtaaga
     2641 tgcgccccgc atgagacggc atgccgtctc tagagctaga aatagcaagt taaaataagg
     2701 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta attcgaaaag
     2761 cctgctcaac gagcaggctt ttttggtcga cagttcatag gtgattgcgg atcccgtcgt
     2821 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
     2881 aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc
     2941 ggtcgagttc tggaccgacc ggctcgggtt ctcccgcgac ttcgtggagg acgacttcgc
     3001 cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc
     3061 ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc
     3121 ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga
     3181 gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt
     3241 ggccgaggag caggactgag agctcgcttg gactcctgtt gatagatcca gtaatgacct
     3301 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     3361 agaatccaag cactagctag taacaactta tatcgtatgg ggctgacttc aggtgctaca
     3421 tttgaagaga taaat
//
