LOCUS       62056_20320        3323 bp DNA     circular SYN 19-APR-2020
DEFINITION  Cloning vector pSyn_Mz_Mj_sfGFP_190-200(dest), complete sequence.
ACCESSION   MN882187
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3323)
  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 3323)
  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 3323)
  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..3323
                     /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    1379..1396
                     /label=D190-Y200 Golden Gate destination site
                     /note="D190-Y200 Golden Gate destination site"
     CDS             1495..1518
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     CDS             1555..1578
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     terminator      1632..1679
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      1709..1727
                     /note="promoter for bacteriophage T7 RNA polymerase; T7 
                     promoter"
                     /regulatory_class="promoter"
     protein_bind    1728..1752
                     /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    1764..1768
                     /label=5' proK leader
                     /note="5' proK leader"
     misc_feature    1769..1816
                     /note="tRNA-Pyl; Methanosarcina mazei tRNAPyl(dest)"
     misc_feature    1776..1793
                     /label=tRNAPyl Golden Gate destination site
                     /note="tRNAPyl Golden Gate destination site"
     misc_feature    1817..1855
                     /label=3' proK trailer
                     /note="3' proK trailer"
     regulatory      1856..1903
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase; T7 terminator"
                     /regulatory_class="terminator"
     regulatory      1950..1968
                     /note="promoter for bacteriophage T7 RNA polymerase; T7 
                     promoter"
                     /regulatory_class="promoter"
     protein_bind    1969..1993
                     /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    2005..2009
                     /label=5' proK leader
                     /note="5' proK leader"
     misc_feature    2010..2052
                     /label=Methanocaldococcus jannaschii tRNApAzF(dest)
                     /note="Methanocaldococcus jannaschii tRNApAzF(dest)"
     misc_feature    2020..2037
                     /label=tRNApAzF Golden Gate destination site
                     /note="tRNApAzF Golden Gate destination site"
     misc_feature    2053..2091
                     /label=3' proK trailer
                     /note="3' proK trailer"
     regulatory      2092..2139
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase; T7 terminator"
                     /regulatory_class="terminator"
     regulatory      2177..2283
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2304..2379
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2387..2422
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     regulatory      2435..2540
                     /label=proK promoter
                     /note="proK promoter"
                     /regulatory_class="promoter"
     misc_RNA        2559..2630
                     /note="guide RNA scaffold for the CRISPR/Cas9 system; gRNA 
                     scaffold"
     regulatory      2638..2673
                     /label=proK terminator
                     /note="proK terminator"
                     /regulatory_class="terminator"
     promoter        2707..2754
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             2773..3144
                     /label=BleoR
                     /note="antibiotic-binding protein"
     terminator      3159..3253
                     /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 ctatcagaga
     1381 gaccggtacc ggtctcaaga cccgaacgag aagcgtgatc acatggtgct gcttgaattt
     1441 gttaccgcgg caggtattac acacggcatg gatgagttgt ataagggatc cgcttggagc
     1501 cacccgcagt tcgagaaagg tggaggttcc ggaggtggat cgggaggttc ggcgtggagc
     1561 cacccgcagt tcgaaaaata ataaaagctt aattagctga gcttggactc cctgccaccg
     1621 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1681 tgaaaggagg aactatatcc ggattggtta atacgactca ctatagggga attgtgagcg
     1741 gataacaatt cccctctaga aaagcattgg aaaccgagac cggtaccggt ctcttagatt
     1801 cccggggttt ccgccaaatt cgaaaagcct gctcaacgag caggcttttt tgcatctagc
     1861 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgcctgaac gagcaggctg
     1921 ctgaaaggag gaactatatc cggattggtt aatacgactc actatagggg aattgtgagc
     1981 ggataacaat tcccctctag aaaagcattc cggcggtagg agaccggtac cggtctcccc
     2041 tccgccggac caaattcgaa aagcctgctc aacgagcagg cttttttgca tctagcataa
     2101 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc ctgaacgagc aggctttttt
     2161 gcataagctt cctagtggca gcggctaact aagcggcctg ctgactttct cgccgatcaa
     2221 aaggcatttt gctattaagg gattgacgag ggcgtatctg cgcagtaaga tgcgccccgc
     2281 attggagacg ccatggcgtc tcggttttag agctagaaat agcaagttaa aataaggcta
     2341 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttaatt cgaaaagcct
     2401 gctcaacgag caggcttttt tggtcgacag tagtggcagc ggctaactaa gcggcctgct
     2461 gactttctcg ccgatcaaaa ggcattttgc tattaaggga ttgacgaggg cgtatctgcg
     2521 cagtaagatg cgccccgcat gagacggcat gccgtctcta gagctagaaa tagcaagtta
     2581 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttaat
     2641 tcgaaaagcc tgctcaacga gcaggctttt ttggtcgaca gttcataggt gattgcggat
     2701 cccgtcgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     2761 aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc
     2821 gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgcgactt cgtggaggac
     2881 gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag
     2941 gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc
     3001 gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag
     3061 atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg
     3121 cacttcgtgg ccgaggagca ggactgagag ctcgcttgga ctcctgttga tagatccagt
     3181 aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt
     3241 tattggtgag aatccaagca ctagctagta acaacttata tcgtatgggg ctgacttcag
     3301 gtgctacatt tgaagagata aat
//
