LOCUS       Exported                8899 bp ds-DNA     circular SYN 29-AUG-2018
DEFINITION  CAG promoter expression plasmid for human codon optimized 
            AsCas12a(E174R/S542R) nuclease with C-terminal NLS and HA tag .
ACCESSION   .
VERSION     .
KEYWORDS    SQT1659.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8899)
  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 8899)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 29, 2018 from SnapGene 4.2.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8899
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1776..5696
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          3417..3419
                     /organism="pCAG-CFP"
                     /mol_type="other DNA"
     source          3417..3419
                     /organism="pY010 (pcDNA3.1-hAsCpf1)"
                     /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..5696
                     /product="Engineered CRISPR-associated protein from 
                     Acidaminococcus sp. BV3L6 with residue substitutions 
                     E174R/S542R"
                     /label=Human codon optimized AsCas12a(E174R/S542R)
                     /note="/modified_by=User"
     misc_feature    2295..2297
                     /label=E174R
                     /label=nonstandard type: misc_marker
                     /note="/created_by=User"
     misc_feature    3399..3401
                     /label=S542R
                     /label=nonstandard type: misc_marker
                     /note="/created_by=User"
     regulatory      5416..5425
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             5697..5744
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5751..5831
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_site      5986..6041
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     protein_bind    6426..6442
                     /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(6450..6480)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6495..6516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6575..6770
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6621..6756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6776..6910
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(7149..7737)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7908..8768)
                     /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(8769..8873)
                     /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 ccaccatgac acagttcgag ggctttacca
     1801 acctgtatca ggtgagcaag acactgcggt ttgagctgat cccacagggc aagaccctga
     1861 agcacatcca ggagcagggc ttcatcgagg aggacaaggc ccgcaatgat cactacaagg
     1921 agctgaagcc catcatcgat cggatctaca agacctatgc cgaccagtgc ctgcagctgg
     1981 tgcagctgga ttgggagaac ctgagcgccg ccatcgactc ctatagaaag gagaaaaccg
     2041 aggagacaag gaacgccctg atcgaggagc aggccacata tcgcaatgcc atccacgact
     2101 acttcatcgg ccggacagac aacctgaccg atgccatcaa taagagacac gccgagatct
     2161 acaagggcct gttcaaggcc gagctgttta atggcaaggt gctgaagcag ctgggcaccg
     2221 tgaccacaac cgagcacgag aacgccctgc tgcggagctt cgacaagttt acaacctact
     2281 tctccggctt ttatagaaac aggaagaacg tgttcagcgc cgaggatatc agcacagcca
     2341 tcccacaccg catcgtgcag gacaacttcc ccaagtttaa ggagaattgt cacatcttca
     2401 cacgcctgat caccgccgtg cccagcctgc gggagcactt tgagaacgtg aagaaggcca
     2461 tcggcatctt cgtgagcacc tccatcgagg aggtgttttc cttccctttt tataaccagc
     2521 tgctgacaca gacccagatc gacctgtata accagctgct gggaggaatc tctcgggagg
     2581 caggcaccga gaagatcaag ggcctgaacg aggtgctgaa tctggccatc cagaagaatg
     2641 atgagacagc ccacatcatc gcctccctgc cacacagatt catccccctg tttaagcaga
     2701 tcctgtccga taggaacacc ctgtctttca tcctggagga gtttaagagc gacgaggaag
     2761 tgatccagtc cttctgcaag tacaagacac tgctgagaaa cgagaacgtg ctggagacag
     2821 ccgaggccct gtttaacgag ctgaacagca tcgacctgac acacatcttc atcagccaca
     2881 agaagctgga gacaatcagc agcgccctgt gcgaccactg ggatacactg aggaatgccc
     2941 tgtatgagcg gagaatctcc gagctgacag gcaagatcac caagtctgcc aaggagaagg
     3001 tgcagcgcag cctgaagcac gaggatatca acctgcagga gatcatctct gccgcaggca
     3061 aggagctgag cgaggccttc aagcagaaaa ccagcgagat cctgtcccac gcacacgccg
     3121 ccctggatca gccactgcct acaaccctga agaagcagga ggagaaggag atcctgaagt
     3181 ctcagctgga cagcctgctg ggcctgtacc acctgctgga ctggtttgcc gtggatgagt
     3241 ccaacgaggt ggaccccgag ttctctgccc ggctgaccgg catcaagctg gagatggagc
     3301 cttctctgag cttctacaac aaggccagaa attatgccac caagaagccc tactccgtgg
     3361 agaagttcaa gctgaacttt cagatgccta cactggccag aggctgggac gtgaataagg
     3421 agaagaacaa tggcgccatc ctgtttgtga agaacggcct gtactatctg ggcatcatgc
     3481 caaagcagaa gggcaggtat aaggccctga gcttcgagcc cacagagaaa accagcgagg
     3541 gctttgataa gatgtactat gactacttcc ctgatgccgc caagatgatc ccaaagtgca
     3601 gcacccagct gaaggccgtg acagcccact ttcagaccca cacaaccccc atcctgctgt
     3661 ccaacaattt catcgagcct ctggagatca caaaggagat ctacgacctg aacaatcctg
     3721 agaaggagcc aaagaagttt cagacagcct acgccaagaa aaccggcgac cagaagggct
     3781 acagagaggc cctgtgcaag tggatcgact tcacaaggga ttttctgtcc aagtatacca
     3841 agacaacctc tatcgatctg tctagcctgc ggccatcctc tcagtataag gacctgggcg
     3901 agtactatgc cgagctgaat cccctgctgt accacatcag cttccagaga atcgccgaga
     3961 aggagatcat ggatgccgtg gagacaggca agctgtacct gttccagatc tataacaagg
     4021 actttgccaa gggccaccac ggcaagccta atctgcacac actgtattgg accggcctgt
     4081 tttctccaga gaacctggcc aagacaagca tcaagctgaa tggccaggcc gagctgttct
     4141 accgccctaa gtccaggatg aagaggatgg cacaccggct gggagagaag atgctgaaca
     4201 agaagctgaa ggatcagaaa accccaatcc ccgacaccct gtaccaggag ctgtacgact
     4261 atgtgaatca cagactgtcc cacgacctgt ctgatgaggc cagggccctg ctgcccaacg
     4321 tgatcaccaa ggaggtgtct cacgagatca tcaaggatag gcgctttacc agcgacaagt
     4381 tctttttcca cgtgcctatc acactgaact atcaggccgc caattcccca tctaagttca
     4441 accagagggt gaatgcctac ctgaaggagc accccgagac acctatcatc ggcatcgatc
     4501 ggggcgagag aaacctgatc tatatcacag tgatcgactc caccggcaag atcctggagc
     4561 agcggagcct gaacaccatc cagcagtttg attaccagaa gaagctggac aacagggaga
     4621 aggagagggt ggcagcaagg caggcctggt ctgtggtggg cacaatcaag gatctgaagc
     4681 agggctatct gagccaggtc atccacgaga tcgtggacct gatgatccac taccaggccg
     4741 tggtggtgct ggagaacctg aatttcggct ttaagagcaa gaggaccggc atcgccgaga
     4801 aggccgtgta ccagcagttc gagaagatgc tgatcgataa gctgaattgc ctggtgctga
     4861 aggactatcc agcagagaaa gtgggaggcg tgctgaaccc ataccagctg acagaccagt
     4921 tcacctcctt tgccaagatg ggcacccagt ctggcttcct gttttacgtg cctgccccat
     4981 atacatctaa gatcgatccc ctgaccggct tcgtggaccc cttcgtgtgg aaaaccatca
     5041 agaatcacga gagccgcaag cacttcctgg agggcttcga ctttctgcac tacgacgtga
     5101 aaaccggcga cttcatcctg cactttaaga tgaacagaaa tctgtccttc cagaggggcc
     5161 tgcccggctt tatgcctgca tgggatatcg tgttcgagaa gaacgagaca cagtttgacg
     5221 ccaagggcac ccctttcatc gccggcaaga gaatcgtgcc agtgatcgag aatcacagat
     5281 tcaccggcag ataccgggac ctgtatcctg ccaacgagct gatcgccctg ctggaggaga
     5341 agggcatcgt gttcagggat ggctccaaca tcctgccaaa gctgctggag aatgacgatt
     5401 ctcacgccat cgacaccatg gtggccctga tccgcagcgt gctgcagatg cggaactcca
     5461 atgccgccac aggcgaggac tatatcaaca gccccgtgcg cgatctgaat ggcgtgtgct
     5521 tcgactcccg gtttcagaac ccagagtggc ccatggacgc cgatgccaat ggcgcctacc
     5581 acatcgccct gaagggccag ctgctgctga atcacctgaa ggagagcaag gatctgaagc
     5641 tgcagaacgg catctccaat caggactggc tggcctacat ccaggagctg cgcaacaaaa
     5701 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc tacccatacg
     5761 atgttccaga ttacgcttat ccctacgacg tgcctgatta tgcataccca tatgatgtcc
     5821 ccgactatgc ctaaagcggc cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     5881 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     5941 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     6001 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     6061 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     6121 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     6181 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     6241 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     6301 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     6361 ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     6421 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     6481 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     6541 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     6601 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     6661 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     6721 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     6781 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6841 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     6901 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6961 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     7021 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     7081 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     7141 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     7201 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     7261 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     7321 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     7381 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     7441 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     7501 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     7561 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     7621 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     7681 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7741 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     7801 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     7861 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     7921 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     7981 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     8041 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     8101 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     8161 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     8221 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     8281 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     8341 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     8401 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     8461 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     8521 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     8581 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     8641 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     8701 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     8761 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     8821 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     8881 ccccgaaaag tgccacctg
//
