LOCUS       Exported                8761 bp ds-DNA     circular SYN 29-AUG-2018
DEFINITION  CAG promoter expression plasmid for human codon optimized Mb3Cas12a 
            nuclease with C-terminal NLS and HA tag .
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-hMb3Cas12a-NLS(nucleoplasmin)-3xHA (RTW2500)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8761)
  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 8761)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 29, 2018 from SnapGene 4.2.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8761
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1776..5693
                     /organism="pCAG-CFP"
                     /mol_type="other DNA"
     source          1776..5693
                     /organism="synthetic DNA construct"
                     /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..5558
                     /label=hMb3Cas12a
                     /note="/created_by=User"
     CDS             5559..5606
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5613..5693
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_site      5848..5903
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     protein_bind    6288..6304
                     /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(6312..6342)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6357..6378
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6437..6632
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6483..6618
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6638..6772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(7011..7599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7770..8630)
                     /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(8631..8735)
                     /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 ccaccatgct gttccaggac ttcacccacc
     1801 tgtaccccct gagcaagacc gtcagattcg agctgaagcc catcggcaag accctggaac
     1861 acatccacgc caagaacttt ctgaaccagg acgagacaat ggccgacatg taccagaaag
     1921 tgaaggccat cctggacgac taccaccggg actttatcgc cgacatgatg ggcgaagtga
     1981 agctgaccaa gctggccgag ttctacgacg tgtacctgaa gttccggaag aaccccaagg
     2041 acgacggcct gcagaagcag ctgaaagacc tgcaggctgt gctgcggaaa gaaatcgtga
     2101 agcctatcgg aaacggcggc aagtacaagg ccggctacga cagactgttc ggcgccaagc
     2161 tgttcaagga cggcaaagag ctgggcgacc tggccaagtt cgtgatcgcc caggaaggcg
     2221 agagcagccc taagctggcc cacctggccc atttcgagaa gttcagcacc tacttcaccg
     2281 gcttccacga caaccgcaag aacatgtaca gcgacgagga caagcacacc gccattgcct
     2341 accggctgat ccacgagaac ctgccccggt tcatcgacaa cctgcagatc ctggccacca
     2401 tcaagcagaa gcacagcgcc ctgtacgacc agatcatcaa cgagctgacc gccagcggcc
     2461 tggatgtgtc tctggcctct cacctggatg gctaccacaa gctgctgaca caggaaggca
     2521 tcaccgccta caacaccctg ctgggcggaa tctctggcga ggccggcagc agaaagatcc
     2581 agggcatcaa tgaactgatc aacagccacc acaaccagca ctgccacaag agcgagcgga
     2641 ttgccaagct gcggcccctg cacaagcaga ttctgagcga cggaatgggc gtgtccttcc
     2701 tgcccagcaa gttcgccgac gacagcgaag tgtgccaggc cgtgaatgag ttctaccggc
     2761 actacgccga cgtgttcgcc aaggtgcaga gcctgttcga cggcttcgac gactatcaga
     2821 aagacggcat ctacgtggag tacaagaacc tgaacgagct gtccaagcag gccttcggcg
     2881 acttcgctct gctgggaaga gtgctggacg gctactatgt ggacgtcgtg aaccccgagt
     2941 tcaacgagag atttgccaag gccaagaccg acaacgccaa agccaagctg acaaaagaga
     3001 aggacaagtt catcaagggc gtgcactccc tggcttctct ggaacaggcc atcgagcact
     3061 acaccgccag acacgacgac gagtctgtgc aggccggaaa gctgggccag tacttcaagc
     3121 acggactggc cggcgtggac aaccccatcc agaagatcca caacaaccac tctacaatca
     3181 agggcttcct ggaaagagag cggcctgccg gcgaaagagc cctgcccaag atcaagagcg
     3241 acaagagccc cgagatcaga cagctgaaag agctgctgga caacgccctg aacgtggccc
     3301 acttcgccaa actgctgacc accaagacca cactgcacaa tcaggatggc aacttctacg
     3361 gcgagttcgg agccctgtat gatgagctgg ccaagatcgc caccctgtac aacaaagtgc
     3421 gggactacct gagccagaag cccttcagca ccgagaagta caagctgaat ttcggcaacc
     3481 ctaccctgct gaacggctgg gacctgaaca aagagaaaga taacttcggc gtgatcctgc
     3541 agaaggacgg ctgctactac ctggccctgc tggataaggc ccacaagaag gtgttcgata
     3601 acgcccccaa caccggcaag tctgtgtatc agaagatgat ctataagctg ctgcctggcc
     3661 ccaacaagat gctgcccaag gtgttcttcg ccaagagcaa cctggactac tacaacccca
     3721 gcgccgaact gctggacaaa tacgcccagg gcacacacaa gaaaggcgac aacttcaacc
     3781 tgaaggactg ccacgccctg atcgatttct tcaaggccgg aatcaacaag caccccgagt
     3841 ggcagcactt cggcttcaag ttcagcccca ccagcagcta ccaggacctg agcgacttct
     3901 acagagaggt ggaaccccag ggctaccaag tgaagttcgt ggacatcaac gccgactaca
     3961 tcaatgagct ggtggaacag ggccagctgt acctgtttca gatctacaac aaggacttta
     4021 gccccaaggc ccacggcaag cccaacctgc acaccctgta tttcaaggcc ctgtttagcg
     4081 aggacaacct cgtgaatccc atctacaaac tgaacgggga ggccgagatc ttctaccgga
     4141 aggcctccct ggacatgaac gagacaacca tccacagagc cggcgaggtg ctggaaaaca
     4201 agaaccctga caaccctaag aaacggcagt tcgtgtacga catcatcaag gacaagcggt
     4261 acacccagga taagttcatg ctgcacgtgc ccatcaccat gaacttcgga gtgcagggca
     4321 tgaccatcaa agagttcaac aaaaaagtga accagagcat ccagcagtac gacgaagtga
     4381 acgtgatcgg catcgaccgg ggcgagcggc atctgctgta tctgaccgtg atcaactcca
     4441 agggcgagat tctggaacag agatccctga acgacatcac caccgcctcc gccaacggca
     4501 cccagatgac caccccttac cacaagatcc tggacaagcg cgagatcgag cggctgaacg
     4561 ccagagtggg atggggagag atcgagacaa tcaaagaact gaagtccggc tacctgtccc
     4621 acgtggtgca tcagatctcc cagctgatgc tgaagtacaa cgccatcgtg gtgctggaag
     4681 atctgaattt tggcttcaag aggggccggt tcaaggtgga aaagcagatc taccagaact
     4741 tcgagaatgc cctgatcaag aaactgaacc acctggtgct gaaagacaag gccgacgacg
     4801 agatcggcag ctacaagaac gccctgcagc tgactaacaa cttcaccgat ctgaagagta
     4861 tcggcaagca gaccggcttt ctgttctacg tgcccgcctg gaataccagc aagatcgacc
     4921 ccgagacagg cttcgtggac ctgctgaagc ctagatacga gaatatcgcc cagagccagg
     4981 ccttcttcgg caagttcgac aagatctgct acaacgccga taggggctac ttcgagttcc
     5041 acatcgacta cgccaagttc aatgacaagg ccaaaaacag ccggcagatc tggaaaatct
     5101 gcagccacgg cgataagcgc tacgtgtacg ataagaccgc caaccagaac aagggcgcca
     5161 ccatcggagt gaatgtgaac gatgagctga agtccctgtt cacccgctac cacatcaacg
     5221 ataagcagcc caatctcgtg atggacatct gccagaacaa cgacaaagag tttcacaaga
     5281 gcctgatgta tctgctgaaa acactgctgg ctctgcggta cagcaacgcc agctccgacg
     5341 aggatttcat cctgagcccc gtggccaacg acgagggcgt gttcttcaat agcgccctgg
     5401 ccgacgatac ccagccccag aatgccgatg ccaacggcgc ctaccacatt gccctgaagg
     5461 gactgtggct gctgaacgaa ctgaagaaca gcgacgatct gaacaaagtg aagctggcca
     5521 tcgacaacca gacctggctg aatttcgccc agaacagaaa gaggccggcg gccacgaaga
     5581 aggccggcca ggccaagaag aagaagggat cctacccata cgatgttcca gattacgctt
     5641 atccctacga cgtgcctgat tatgcatacc catatgatgt ccccgactat gcctaaagcg
     5701 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     5761 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     5821 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     5881 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     5941 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     6001 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     6061 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     6121 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     6181 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     6241 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     6301 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     6361 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6421 tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     6481 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     6541 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     6601 gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa
     6661 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     6721 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat
     6781 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6841 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6901 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6961 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     7021 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     7081 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     7141 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     7201 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     7261 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     7321 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     7381 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     7441 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7501 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7561 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7621 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7681 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7741 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7801 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7861 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7921 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7981 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     8041 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     8101 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     8161 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     8221 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     8281 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     8341 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     8401 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     8461 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     8521 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     8581 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8641 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8701 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8761 g
//
