LOCUS       Exported                8878 bp ds-DNA     circular SYN 29-AUG-2018
DEFINITION  Human expression plasmid for FnCas12a-NLS-3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-hFnCas12a-NLS(nucleoplasmin)-3xHA (AAS1472)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8878)
  AUTHORS   Kleinstiver, BP, Tsai, SQ, Prew, MS, Nguyen, NT, Welch, MM, Lopez, 
            JM, McCaw, ZR, Aryee, MJ, Joung, JK
  TITLE     Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human 
            cells.
  JOURNAL   Nat Biotechnol. 2016 Jun 27. doi: 10.1038/nbt.3620.
  PUBMED    27347757
REFERENCE   2  (bases 1 to 8878)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 29, 2018 from SnapGene 4.2.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8878
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1776..5675
                     /organism="pCAG-CFP"
                     /mol_type="other DNA"
     source          1776..5675
                     /organism="pY004 (pcDNA3.1-hFnCpf1)"
                     /mol_type="other DNA"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..660
                     /label=chicken beta-actin promoter
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     CDS             1776..5675
                     /label=Human codon optimized FnCas12a
                     /note="/modified_by=User"
                     /Product="FnCas12a (humanized) Francisella tularensis 
                     subsp. novicida U112"
     CDS             5676..5723
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5730..5810
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_site      5965..6020
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     protein_bind    6405..6421
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     promoter        complement(6429..6459)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6474..6495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6554..6749
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6600..6735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6755..6889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(7128..7716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7887..8747)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8748..8852)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt ctgcagtcga
     1741 cggtaccgcg ggcccgggat ccaccggtcg ccaccatgag catctaccag gagttcgtca
     1801 acaagtattc actgagtaag acactgcggt tcgagctgat cccacagggc aagacactgg
     1861 agaacatcaa ggcccgaggc ctgattctgg acgatgagaa gcgggcaaaa gactataaga
     1921 aagccaagca gatcattgat aaataccacc agttctttat cgaggaaatt ctgagctccg
     1981 tgtgcatcag tgaggatctg ctgcagaatt actcagacgt gtacttcaag ctgaagaaga
     2041 gcgacgatga caacctgcag aaggacttca agtccgccaa ggacaccatc aagaaacaga
     2101 ttagcgagta catcaaggac tccgaaaagt ttaaaaatct gttcaaccag aatctgatcg
     2161 atgctaagaa aggccaggag tccgacctga tcctgtggct gaaacagtct aaggacaatg
     2221 ggattgaact gttcaaggct aactccgata tcactgatat tgacgaggca ctggaaatca
     2281 tcaagagctt caagggatgg accacatact ttaaaggctt ccacgagaac cgcaagaacg
     2341 tgtactccag caacgacatt cctacctcca tcatctaccg aatcgtcgat gacaatctgc
     2401 caaagttcct ggagaacaag gccaaatatg aatctctgaa ggacaaagct cccgaggcaa
     2461 ttaattacga acagatcaag aaagatctgg ctgaggaact gacattcgat atcgactata
     2521 agactagcga ggtgaaccag agggtctttt ccctggacga ggtgtttgaa atcgccaatt
     2581 tcaacaatta cctgaaccag tccggcatta ctaaattcaa taccatcatt ggcgggaagt
     2641 ttgtgaacgg ggagaatacc aagcgcaagg gaattaacga atacatcaat ctgtatagcc
     2701 agcagatcaa cgacaaaact ctgaagaaat acaagatgtc tgtgctgttc aaacagatcc
     2761 tgagtgatac cgagtccaag tcttttgtca ttgataaact ggaagatgac tcagacgtgg
     2821 tcactaccat gcagagcttt tatgagcaga tcgccgcttt caagacagtg gaggaaaaat
     2881 ctattaagga aactctgagt ctgctgttcg atgacctgaa agcccagaag ctggacctga
     2941 gtaagatcta cttcaaaaac gataagagtc tgacagacct gtcacagcag gtgtttgatg
     3001 actattccgt gattgggacc gccgtcctgg agtacattac acagcagatc gctccaaaga
     3061 acctggataa tccctctaag aaagagcagg aactgatcgc taagaaaacc gagaaggcaa
     3121 aatatctgag tctggaaaca attaagctgg cactggagga gttcaacaag cacagggata
     3181 ttgacaaaca gtgccgcttt gaggaaatcc tggccaactt cgcagccatc cccatgattt
     3241 ttgatgagat cgcccagaac aaagacaatc tggctcagat cagtattaag taccagaacc
     3301 agggcaagaa agacctgctg caggcttcag cagaagatga cgtgaaagcc atcaaggatc
     3361 tgctggacca gaccaacaat ctgctgcaca agctgaaaat cttccatatt agtcagtcag
     3421 aggataaggc taatatcctg gataaagacg aacacttcta cctggtgttc gaggaatgtt
     3481 acttcgagct ggcaaacatt gtccccctgt ataacaagat taggaactac atcacacaga
     3541 agccttactc tgacgagaag tttaaactga acttcgaaaa tagtaccctg gccaacgggt
     3601 gggataagaa caaggagcct gacaacacag ctatcctgtt catcaaggat gacaagtact
     3661 atctgggagt gatgaataag aaaaacaata agatcttcga tgacaaagcc attaaggaga
     3721 acaaagggga aggatacaag aaaatcgtgt ataagctgct gcccggcgca aataagatgc
     3781 tgcctaaggt gttcttcagc gccaagagta tcaaattcta caacccatcc gaggacatcc
     3841 tgcggattag aaatcactca acacatacta agaacgggag cccccagaag ggatatgaga
     3901 aatttgagtt caacatcgag gattgcagga agtttattga cttctacaag cagagcatct
     3961 ccaaacaccc tgaatggaag gattttggct tccggttttc cgacacacag agatataact
     4021 ctatcgacga gttctaccgc gaggtggaaa atcaggggta taagctgact tttgagaaca
     4081 tttctgaaag ttacatcgac agcgtggtca atcagggaaa gctgtacctg ttccagatct
     4141 ataacaaaga tttttcagca tacagcaagg gcagaccaaa cctgcataca ctgtactgga
     4201 aggccctgtt cgatgagagg aatctgcagg acgtggtcta taaactgaac ggagaggccg
     4261 aactgtttta ccggaagcag tctattccta agaaaatcac tcacccagct aaggaggcca
     4321 tcgctaacaa gaacaaggac aatcctaaga aagagagcgt gttcgaatac gatctgatta
     4381 aggacaagcg gttcaccgaa gataagttct ttttccattg tccaatcacc attaacttca
     4441 agtcaagcgg cgctaacaag ttcaacgacg agatcaatct gctgctgaag gaaaaagcaa
     4501 acgatgtgca catcctgagc attgaccgag gagagcggca tctggcctac tataccctgg
     4561 tggatggcaa agggaatatc attaagcagg atacattcaa catcattggc aatgaccgga
     4621 tgaaaaccaa ctaccacgat aaactggctg caatcgagaa ggatagagac tcagctagga
     4681 aggactggaa gaaaatcaac aacattaagg agatgaagga aggctatctg agccaggtgg
     4741 tccatgagat tgcaaagctg gtcatcgaat acaatgccat tgtggtgttc gaggatctga
     4801 acttcggctt taagaggggg cgctttaagg tggaaaaaca ggtctatcag aagctggaga
     4861 aaatgctgat cgaaaagctg aattacctgg tgtttaaaga taacgagttc gacaagaccg
     4921 gaggcgtcct gagagcctac cagctgacag ctccctttga aactttcaag aaaatgggaa
     4981 aacagacagg catcatctac tatgtgccag ccggattcac ttccaagatc tgccccgtga
     5041 ccggctttgt caaccagctg taccctaaat atgagtcagt gagcaagtcc caggaatttt
     5101 tcagcaagtt cgataagatc tgttataatc tggacaaggg gtacttcgag ttttccttcg
     5161 attacaagaa cttcggcgac aaggccgcta aggggaaatg gaccattgcc tccttcggat
     5221 ctcgcctgat caactttcga aattccgata aaaaccacaa ttgggacact agggaggtgt
     5281 acccaaccaa ggagctggaa aagctgctga aagactactc tatcgagtat ggacatggcg
     5341 aatgcatcaa ggcagccatc tgtggcgaga gtgataagaa atttttcgcc aagctgacct
     5401 cagtgctgaa tacaatcctg cagatgcgga actcaaagac cgggacagaa ctggactatc
     5461 tgattagccc cgtggctgat gtcaacggaa acttcttcga cagcagacag gcacccaaaa
     5521 atatgcctca ggatgcagac gccaacgggg cctaccacat cgggctgaag ggactgatgc
     5581 tgctgggccg gatcaagaac aatcaggagg ggaagaagct gaacctggtc attaagaacg
     5641 aggaatactt cgagtttgtc cagaatagaa ataacaaaag gccggcggcc acgaaaaagg
     5701 ccggccaggc aaaaaagaaa aagggatcct acccatacga tgttccagat tacgcttatc
     5761 cctacgacgt gcctgattat gcatacccat atgatgtccc cgactatgcc taaagcggcc
     5821 gcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg
     5881 ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     5941 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     6001 ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc
     6061 agaatgagta tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca
     6121 aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     6181 tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt
     6241 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     6301 cctgactact cccagtcata gctgtccctc ttctcttatg gagatccctc gacctgcagc
     6361 ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     6421 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6481 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6541 tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     6601 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     6661 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     6721 gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg
     6781 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     6841 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa
     6901 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     6961 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     7021 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     7081 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     7141 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     7201 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     7261 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     7321 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     7381 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     7441 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     7501 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     7561 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     7621 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     7681 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7741 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7801 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7861 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7921 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7981 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     8041 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     8101 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     8161 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     8221 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     8281 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     8341 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     8401 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     8461 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     8521 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     8581 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     8641 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8701 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8761 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8821 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg
//
