LOCUS       V000880                 7374 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V000880
VERSION     V000880
KEYWORDS    pC0073 EiCsm6  His6-TwinStrep-SUMO-BsaI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7374)
  AUTHORS   Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang
            F
  TITLE     Multiplexed and portable nucleic acid detection platform with Cas13,
            Cas12a, and Csm6.
  JOURNAL   Science. 2018 Apr 27;360(6387):439-444. doi:
            10.1126/science.aaq0179. Epub 2018 Feb 15.
   PUBMED   29449508
REFERENCE   2  (bases 1 to 7374)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7374)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1126/science.aaq0179"; journalName: "Science"; date:
            "2018-04-27- 27"; volume: "360"; issue: "6387"; pages: "439-444"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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)."
     RBS             40..62
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /label="6xHis"
                     /note="6xHis affinity tag"
     CDS             108..125
                     /label="thrombin site"
                     /note="thrombin recognition and cleavage site"
     CDS             138..221
                     /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)"
     CDS             222..515
                     /label="SUMO"
                     /note="cleavable ubiquitin-like protein tag"
     CDS             519..1808
                     /gene="csm6"
                     /label="CRISPR system endoribonuclease Csm6"
                     /note="CRISPR system endoribonuclease Csm6 from
                     Enterococcus italicus (strain DSM 15952 / CCUG 50447 / LMG
                     22039 / TP 1.5). Accession#: E6LHV2"
     terminator      1870..1917
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     promoter        complement(2092..2120)
                     /label="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     primer_bind     complement(2147..2165)
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        2233..2337
                     /label="AmpR promoter"
     CDS             2338..3195
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      3369..3957
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     4111..4128
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     misc_feature    complement(4143..4282)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     primer_bind     4368..4390
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4387..4575)
                     /label="rop"
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(5853..5874)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5890..6969)
                     /label="lacI"
                     /note="lac repressor"
     promoter        complement(6970..7047)
                     /label="lacI promoter"
     primer_bind     7253..7272
                     /label="pBRrevBam"
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     promoter        7356..7374
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      121 gcagccatat ggctagctgg agccatccgc agtttgaaaa aggtggtggt agcggtggtg
      181 gttcaggtgg tagtgcatgg tcacaccctc agtttgagaa aatgtcggac tcagaagtca
      241 atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac atcaatttaa
      301 aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact cctttaagaa
      361 ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta agattcttgt
      421 acgacggtat tagaatccaa gctgatcaga cccctgaaga tttggacatg gaggataacg
      481 atattattga ggctcacaga gaacagattg gtggatctat gaagattctt tttagcccga
      541 ttggcaacac agatccttgg cgcaatgacc gcgatggtgc gatgttgcac atcgttcgtc
      601 actaccaacc tgaccgtgta gtgctttttt ttaccgagtc catctggcaa gggaatcaac
      661 acttctcagg acaacaagcc tttgattggg tgaaaatcat tcagagtatt aacgagaact
      721 gtcagattga aattaaatgt gatactattg aggtggagaa tgatttcgat gcctacaaag
      781 atttgttcca tcagtactta gtcgaggaaa aacgtaaata tcctaatgct gaaattttcc
      841 tgaatgtcac aagcgggact ccgcagatgg aaacaacttt gtgccttgaa tatgtgactt
      901 accccgacaa aatgcgctgc attcaggtca gcaccccgct taagactagc aatgctaaaa
      961 ccaaatacgc gcaagccgat tgccaggaag ttgaccttga aatcgttaat gaagaggagt
     1021 ctcagcaacc ttcacgctgt cacaagatcg ctattctgag ctttcgtgaa gcaatcgtac
     1081 gtaatcagat caaatcatta cttgataatt acgactacga agccgcgctg cagcttgttg
     1141 catcacaaaa gagtttccgc aatgggaagg agattcgcaa aaagctgaaa gagctgatcg
     1201 acgacattaa gatgcaccgt gtgttctcat acttaatcaa acagtatccc cgcaacgaga
     1261 agttgcaaaa agctctgtta cacaccattc ttttagaaat gcgtcaccaa cgtggagaca
     1321 ttgccgaaac tctgatccgt gtaaagagta ttgcagagta tatcgtggaa caatatatcc
     1381 aaaagaacta tccatatctg atcatctaca aagaagacaa gccttacttc aacgttagct
     1441 acagtcaaga gctgaccgag agttatttgg ccctgatgga cagccgcaac aagaagacta
     1501 acaagaaaat gaccgtggac tccctggatc gtatcttggg gtttcccgct taccgcgatt
     1561 tcttgcaact gttagaagcc agcaacgaga tgactaacga gatgaataaa gtaaatgaaa
     1621 ttaataacct tcgcaataag gtcgcccata acttagacag tcttaattta gatcgtgaca
     1681 agaatggtcg caaaatcacg aatgcggtaa ctgcggtgcg caccatgctg ttggcggtct
     1741 tcccagaggt tcaggaaaac gacttccatt atttaaagca atttaatcag agcattaaag
     1801 aacttttgta aggccgcact cgaggcccga aaggaagctg agttggctgc tgccaccgct
     1861 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1921 aaaggaggaa ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     1981 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     2041 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     2101 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
     2161 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2221 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2281 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2341 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2401 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2461 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2521 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2581 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2641 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2701 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2761 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2821 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2881 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2941 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3001 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtcacgc
     3061 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3121 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3181 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3241 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3301 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3361 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3421 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3481 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3541 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3601 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3661 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3721 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3781 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3841 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3901 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3961 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4021 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4081 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4141 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4201 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4261 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4321 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4381 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4441 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4501 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4561 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4621 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4681 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4741 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4801 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4861 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4921 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4981 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5041 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     5101 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     5161 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     5221 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     5281 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     5341 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     5401 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     5461 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     5521 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     5581 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5641 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5701 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5761 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5821 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5881 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5941 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     6001 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     6061 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     6121 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     6181 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6241 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6301 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6361 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6421 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6481 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6541 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6601 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6661 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6721 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6781 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6841 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6901 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6961 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     7021 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     7081 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     7141 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     7201 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     7261 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     7321 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta tagg
//
