LOCUS       6-His-MBP-TEV-Fn       10685 bp DNA     circular SYN 04-JUL-2018
DEFINITION  Bacterial expression plasmid for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    6-His-MBP-TEV-FnCpf1.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10685)
  AUTHORS   Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, 
            Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin 
            EV, Zhang F
  TITLE     Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas 
            System.
  JOURNAL   Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 
            10.1016/j.cell.2015.09.038.
  PUBMED    26422227
REFERENCE   2  (bases 1 to 10685)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10685)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2015 
            Sep 23. pii: S0092-8674(15)01200-3. doi: 
            10.1016/j.cell.2015.09.038."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      532..1120
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(1306..1445)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(1550..1738)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(2513..2534)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2550..3629)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(3630..3707)
                     /label=lacI promoter
     promoter        4016..4034
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4035..4059
                     /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             4074..4096
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4110..4127
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             4128..5225
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             5244..5261
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             5268..5288
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             5289..9188
                     /gene="cas12a"
                     /label=cas12a
                     /note="CRISPR-associated endonuclease Cas12a from
                     Francisella tularensis subsp. novicida (strain U112). 
                     Accession#: A0Q7Q2"
     CDS             9189..9236
                     /label=nucleoplasmin NLS
                     /note="bipartite nuclear localization signal from
                     nucleoplasmin"
     CDS             9243..9323
                     /label=3xHA
                     /note="three tandem HA epitope tags"
     CDS             9348..9371
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             9372..9416
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             9432..9449
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      9516..9563
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      9600..9980
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        10081..10185
                     /label=AmpR promoter
     CDS             join(10186..10685,1..358)
                     /label=AmpR
                     /note="beta-lactamase"
ORIGIN
        1 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
       61 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      121 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      181 gtttattgct gataaatctg gagccggtga gcgtggtagt cgcggtatca ttgcagcact
      241 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      301 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
      361 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      661 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     1321 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     1381 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     1441 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     1501 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     1561 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     1621 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     1681 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     1741 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     1801 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     1861 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     1921 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     1981 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     2041 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     2101 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     2161 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     2221 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     2281 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     2341 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     2401 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     2461 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     2521 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2581 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     2641 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     2701 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     2761 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     2821 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     2881 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     2941 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     3001 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     3061 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     3121 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     3181 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     3241 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     3301 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     3361 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     3421 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     3481 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     3541 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     3601 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     3661 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     3721 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     3781 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     3841 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     3901 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     3961 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     4021 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     4081 aactttaaga aggagatata catatgggtc atcatcatca ccatcacaaa atcgaagaag
     4141 gcaaactggt tatttggatc aatggcgata aaggctataa tggtctggca gaagttggca
     4201 aaaaattcga aaaagatacc ggcattaaag tgaccgttga acatccggat aaactggaag
     4261 aaaaatttcc gcaggttgca gcaaccggtg atggtccgga tattatcttt tgggcacatg
     4321 atcgttttgg tggttatgca cagagcggtc tgctggcaga aattacaccg gataaagcat
     4381 ttcaggacaa actgtatccg tttacctggg atgcagttcg ctataacggt aaactgattg
     4441 catatccgat tgcagttgaa gcactgagcc tgatctataa caaagatctg ctgccgaatc
     4501 cgcctaaaac ctgggaagaa attccggcac tggataaaga actgaaagca aaaggtaaaa
     4561 gcgcactgat gtttaatctg caagaaccgt attttacctg gcctctgatt gcagcagatg
     4621 gtggctatgc attcaaatat gaaaacggca aatatgatat caaagacgtg ggtgttgata
     4681 atgcgggtgc aaaagccggt ctgacctttc tggttgatct gattaaaaac aaacacatga
     4741 acgccgatac cgattatagc attgcagaag cagcatttaa caaaggtgaa accgcaatga
     4801 caattaatgg tccgtgggca tggtcaaata ttgataccag caaagtgaat tatggtgtta
     4861 ccgttctgcc gacatttaaa ggtcagccga gcaaaccgtt tgttggtgtt ctgagcgcag
     4921 gtattaatgc agcaagcccg aacaaagaac tggcaaaaga atttctggaa aactatctgc
     4981 tgaccgatga aggtctggaa gcagtgaata aagataaacc gctgggtgca gttgcactga
     5041 aaagctatga agaagaactg gttaaagatc cgcgtattgc agccaccatg gaaaatgcac
     5101 agaaaggcga aattatgccg aatattccgc agatgagcgc attttggtat gccgttcgta
     5161 ccgcagtgat taatgccgca tcaggtcgtc agaccgttga tgaagccctg aaagatgccc
     5221 agaccggtgc aagcaccgga tccctggttc cgcgtggtag cgctagcgaa aatctgtatt
     5281 ttcagggaat gagcatctac caggagttcg tcaacaagta ttcactgagt aagacactgc
     5341 ggttcgagct gatcccacag ggcaagacac tggagaacat caaggcccga ggcctgattc
     5401 tggacgatga gaagcgggca aaagactata agaaagccaa gcagatcatt gataaatacc
     5461 accagttctt tatcgaggaa attctgagct ccgtgtgcat cagtgaggat ctgctgcaga
     5521 attactcaga cgtgtacttc aagctgaaga agagcgacga tgacaacctg cagaaggact
     5581 tcaagtccgc caaggacacc atcaagaaac agattagcga gtacatcaag gactccgaaa
     5641 agtttaaaaa tctgttcaac cagaatctga tcgatgctaa gaaaggccag gagtccgacc
     5701 tgatcctgtg gctgaaacag tctaaggaca atgggattga actgttcaag gctaactccg
     5761 atatcactga tattgacgag gcactggaaa tcatcaagag cttcaaggga tggaccacat
     5821 actttaaagg cttccacgag aaccgcaaga acgtgtactc cagcaacgac attcctacct
     5881 ccatcatcta ccgaatcgtc gatgacaatc tgccaaagtt cctggagaac aaggccaaat
     5941 atgaatctct gaaggacaaa gctcccgagg caattaatta cgaacagatc aagaaagatc
     6001 tggctgagga actgacattc gatatcgact ataagactag cgaggtgaac cagagggtct
     6061 tttccctgga cgaggtgttt gaaatcgcca atttcaacaa ttacctgaac cagtccggca
     6121 ttactaaatt caataccatc attggcggga agtttgtgaa cggggagaat accaagcgca
     6181 agggaattaa cgaatacatc aatctgtata gccagcagat caacgacaaa actctgaaga
     6241 aatacaagat gtctgtgctg ttcaaacaga tcctgagtga taccgagtcc aagtcttttg
     6301 tcattgataa actggaagat gactcagacg tggtcactac catgcagagc ttttatgagc
     6361 agatcgccgc tttcaagaca gtggaggaaa aatctattaa ggaaactctg agtctgctgt
     6421 tcgatgacct gaaagcccag aagctggacc tgagtaagat ctacttcaaa aacgataaga
     6481 gtctgacaga cctgtcacag caggtgtttg atgactattc cgtgattggg accgccgtcc
     6541 tggagtacat tacacagcag atcgctccaa agaacctgga taatccctct aagaaagagc
     6601 aggaactgat cgctaagaaa accgagaagg caaaatatct gagtctggaa acaattaagc
     6661 tggcactgga ggagttcaac aagcacaggg atattgacaa acagtgccgc tttgaggaaa
     6721 tcctggccaa cttcgcagcc atccccatga tttttgatga gatcgcccag aacaaagaca
     6781 atctggctca gatcagtatt aagtaccaga accagggcaa gaaagacctg ctgcaggctt
     6841 cagcagaaga tgacgtgaaa gccatcaagg atctgctgga ccagaccaac aatctgctgc
     6901 acaagctgaa aatcttccat attagtcagt cagaggataa ggctaatatc ctggataaag
     6961 acgaacactt ctacctggtg ttcgaggaat gttacttcga gctggcaaac attgtccccc
     7021 tgtataacaa gattaggaac tacatcacac agaagcctta ctctgacgag aagtttaaac
     7081 tgaacttcga aaatagtacc ctggccaacg ggtgggataa gaacaaggag cctgacaaca
     7141 cagctatcct gttcatcaag gatgacaagt actatctggg agtgatgaat aagaaaaaca
     7201 ataagatctt cgatgacaaa gccattaagg agaacaaagg ggaaggatac aagaaaatcg
     7261 tgtataagct gctgcccggc gcaaataaga tgctgcctaa ggtgttcttc agcgccaaga
     7321 gtatcaaatt ctacaaccca tccgaggaca tcctgcggat tagaaatcac tcaacacata
     7381 ctaagaacgg gagcccccag aagggatatg agaaatttga gttcaacatc gaggattgca
     7441 ggaagtttat tgacttctac aagcagagca tctccaaaca ccctgaatgg aaggattttg
     7501 gcttccggtt ttccgacaca cagagatata actctatcga cgagttctac cgcgaggtgg
     7561 aaaatcaggg gtataagctg acttttgaga acatttctga aagttacatc gacagcgtgg
     7621 tcaatcaggg aaagctgtac ctgttccaga tctataacaa agatttttca gcatacagca
     7681 agggcagacc aaacctgcat acactgtact ggaaggccct gttcgatgag aggaatctgc
     7741 aggacgtggt ctataaactg aacggagagg ccgaactgtt ttaccggaag cagtctattc
     7801 ctaagaaaat cactcaccca gctaaggagg ccatcgctaa caagaacaag gacaatccta
     7861 agaaagagag cgtgttcgaa tacgatctga ttaaggacaa gcggttcacc gaagataagt
     7921 tctttttcca ttgtccaatc accattaact tcaagtcaag cggcgctaac aagttcaacg
     7981 acgagatcaa tctgctgctg aaggaaaaag caaacgatgt gcacatcctg agcattgacc
     8041 gaggagagcg gcatctggcc tactataccc tggtggatgg caaagggaat atcattaagc
     8101 aggatacatt caacatcatt ggcaatgacc ggatgaaaac caactaccac gataaactgg
     8161 ctgcaatcga gaaggataga gactcagcta ggaaggactg gaagaaaatc aacaacatta
     8221 aggagatgaa ggaaggctat ctgagccagg tggtccatga gattgcaaag ctggtcatcg
     8281 aatacaatgc cattgtggtg ttcgaggatc tgaacttcgg ctttaagagg gggcgcttta
     8341 aggtggaaaa acaggtctat cagaagctgg agaaaatgct gatcgaaaag ctgaattacc
     8401 tggtgtttaa agataacgag ttcgacaaga ccggaggcgt cctgagagcc taccagctga
     8461 cagctccctt tgaaactttc aagaaaatgg gaaaacagac aggcatcatc tactatgtgc
     8521 cagccggatt cacttccaag atctgccccg tgaccggctt tgtcaaccag ctgtacccta
     8581 aatatgagtc agtgagcaag tcccaggaat ttttcagcaa gttcgataag atctgttata
     8641 atctggacaa ggggtacttc gagttttcct tcgattacaa gaacttcggc gacaaggccg
     8701 ctaaggggaa atggaccatt gcctccttcg gatctcgcct gatcaacttt cgaaattccg
     8761 ataaaaacca caattgggac actagggagg tgtacccaac caaggagctg gaaaagctgc
     8821 tgaaagacta ctctatcgag tatggacatg gcgaatgcat caaggcagcc atctgtggcg
     8881 agagtgataa gaaatttttc gccaagctga cctcagtgct gaatacaatc ctgcagatgc
     8941 ggaactcaaa gaccgggaca gaactggact atctgattag ccccgtggct gatgtcaacg
     9001 gaaacttctt cgacagcaga caggcaccca aaaatatgcc tcaggatgca gacgccaacg
     9061 gggcctacca catcgggctg aagggactga tgctgctggg ccggatcaag aacaatcagg
     9121 aggggaagaa gctgaacctg gtcattaaga acgaggaata cttcgagttt gtccagaata
     9181 gaaataacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat
     9241 cctacccata cgatgttcca gattacgctt atccctacga cgtgcctgat tatgcatacc
     9301 catatgatgt ccccgactat gcctaaagcc tcgagggtgg tggtagcgat tataaagatg
     9361 atgatgataa aggcctgaac gatatctttg aagcccagaa aattgaatgg cacgagtaaa
     9421 agcttgtcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     9481 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     9541 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     9601 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     9661 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     9721 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     9781 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     9841 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     9901 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     9961 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
    10021 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
    10081 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
    10141 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
    10201 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
    10261 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
    10321 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
    10381 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
    10441 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
    10501 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
    10561 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
    10621 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
    10681 gagct
//
