LOCUS       Exported                6307 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Level0 Golden Gate part: CDS, AsCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    pEPOR0SP0008
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6307)
  AUTHORS   Raitskin O, Schudoma C, West A, Patron NJ
  TITLE     Comparison of efficiency and specificity of CRISPR-associated (Cas) 
            nucleases in plants: An expanded toolkit for precision genome 
            engineering.
  JOURNAL   PLoS One. 2019 Feb 27;14(2):e0211598. doi: 
            10.1371/journal.pone.0211598. eCollection 2019.
  PUBMED    30811422
REFERENCE   2  (bases 1 to 6307)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6307
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(24..42)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        110..214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             434..1225
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1396..1984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1885..1904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2138..2155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2233..6153
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND
                     HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN
                     AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD
                     KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF
                     ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
                     NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL
                     RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG
                     KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
                     KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
                     RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK
                     ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP
                     LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID
                     LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
                     HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK
                     DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF
                     HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR
                     SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV
                     VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
                     FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
                     VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
                     HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
                     RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
                     KDLKLQNGISNQDWLAYIQELRN"
     regulatory      5873..5882
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             6154..6201
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6208..6288
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
ORIGIN
        1 gtggctcttc agtggacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
       61 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      121 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      181 ataaatgctt caataatatt gaaaaaggaa gagtatgcgc tcacgcaact ggtccagaac
      241 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
      301 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
      361 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      421 aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
      481 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
      541 cgcagtggat ggcggcctga agccacacag cgatattgat ttgctggtta cggtgaccgt
      601 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
      661 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
      721 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
      781 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
      841 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
      901 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
      961 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     1021 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     1081 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     1141 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccattacg taaaaggcga
     1201 gatcaccaag gtagtcggca aataactgtc agaccaagtt tactcatata tactttagat
     1261 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1321 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1381 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1441 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1501 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1561 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1621 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1681 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1741 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1801 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1861 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1921 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1981 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2041 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2101 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2161 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatcact
     2221 ctgtggtctc aaatgacaca gttcgagggc tttaccaacc tgtatcaggt gagcaagaca
     2281 ctgcggtttg agctgatccc acagggcaag accctgaagc acatccagga gcagggcttc
     2341 atcgaggagg acaaggcccg caatgatcac tacaaggagc tgaagcccat catcgatcgg
     2401 atctacaaga cctatgccga ccagtgcctg cagctggtgc agctggattg ggagaacctg
     2461 agcgccgcca tcgactccta tagaaaggag aaaaccgagg agacaaggaa cgccctgatc
     2521 gaggagcagg ccacatatcg caatgccatc cacgactact tcatcggccg gacagacaac
     2581 ctgaccgatg ccatcaataa gagacacgcc gagatctaca agggcctgtt caaggccgag
     2641 ctgtttaatg gcaaggtgct gaagcagctg ggcaccgtga ccacaaccga gcacgagaac
     2701 gccctgctgc ggagcttcga caagtttaca acctacttct ccggctttta tgagaacagg
     2761 aagaacgtgt tcagcgccga ggatatcagc acagccatcc cacaccgcat cgtgcaggac
     2821 aacttcccca agtttaagga gaattgtcac atcttcacac gcctgatcac cgccgtgccc
     2881 agcctgcggg agcactttga gaacgtgaag aaggccatcg gcatcttcgt gagcacctcc
     2941 atcgaggagg tgttttcctt ccctttttat aaccagctgc tgacacagac ccagatcgac
     3001 ctgtataacc agctgctggg aggaatctct cgggaggcag gcaccgagaa gatcaagggc
     3061 ctgaacgagg tgctgaatct ggccatccag aagaatgatg agacagccca catcatcgcc
     3121 tccctgccac acagattcat ccccctgttt aagcagatcc tgtccgatag gaacaccctg
     3181 tctttcatcc tggaggagtt taagagcgac gaggaagtga tccagtcctt ctgcaagtac
     3241 aagacactgc tgagaaacga gaacgtgctg gagacagccg aggccctgtt taacgagctg
     3301 aacagcatcg acctgacaca catcttcatc agccacaaga agctggagac aatcagcagc
     3361 gccctgtgcg accactggga tacactgagg aatgccctgt atgagcggag aatctccgag
     3421 ctgacaggca agatcaccaa gtctgccaag gagaaggtgc agcgcagcct gaagcacgag
     3481 gatatcaacc tgcaggagat catctctgcc gcaggcaagg agctgagcga ggccttcaag
     3541 cagaaaacca gcgagatcct gtcccacgca cacgccgccc tggatcagcc actgcctaca
     3601 accctgaaga agcaggagga gaaggagatc ctgaagtctc agctggacag cctgctgggc
     3661 ctgtaccacc tgctggactg gtttgccgtg gatgagtcca acgaggtgga ccccgagttc
     3721 tctgcccggc tgaccggcat caagctggag atggagcctt ctctgagctt ctacaacaag
     3781 gccagaaatt atgccaccaa gaagccctac tccgtggaga agttcaagct gaactttcag
     3841 atgcctacac tggcctctgg ctgggacgtg aataaggaga agaacaatgg cgccatcctg
     3901 tttgtgaaga acggcctgta ctatctgggc atcatgccaa agcagaaggg caggtataag
     3961 gccctgagct tcgagcccac agagaaaacc agcgagggct ttgataagat gtactatgac
     4021 tacttccctg atgccgccaa gatgatccca aagtgcagca cccagctgaa ggccgtgaca
     4081 gcccactttc agacccacac aacccccatc ctgctgtcca acaatttcat cgagcctctg
     4141 gagatcacaa aggagatcta cgacctgaac aatcctgaga aggagccaaa gaagtttcag
     4201 acagcctacg ccaagaaaac cggcgaccag aagggctaca gagaggccct gtgcaagtgg
     4261 atcgacttca caagggattt tctgtccaag tataccaaga caacctctat cgatctgtct
     4321 agcctgcggc catcctctca gtataaggac ctgggcgagt actatgccga gctgaatccc
     4381 ctgctgtacc acatcagctt ccagagaatc gccgagaagg agatcatgga tgccgtggag
     4441 acaggcaagc tgtacctgtt ccagatctat aacaaggact ttgccaaggg ccaccacggc
     4501 aagcctaatc tgcacacact gtattggacc ggcctgtttt ctccagagaa cctggccaag
     4561 acaagcatca agctgaatgg ccaggccgag ctgttctacc gccctaagtc caggatgaag
     4621 aggatggcac accggctggg agagaagatg ctgaacaaga agctgaagga tcagaaaacc
     4681 ccaatccccg acaccctgta ccaggagctg tacgactatg tgaatcacag actgtcccac
     4741 gacctgtctg atgaggccag ggccctgctg cccaacgtga tcaccaagga ggtgtctcac
     4801 gagatcatca aggataggcg ctttaccagc gacaagttct ttttccacgt gcctatcaca
     4861 ctgaactatc aggccgccaa ttccccatct aagttcaacc agagggtgaa tgcctacctg
     4921 aaggagcacc ccgagacacc tatcatcggc atcgatcggg gcgagagaaa cctgatctat
     4981 atcacagtga tcgactccac cggcaagatc ctggagcagc ggagcctgaa caccatccag
     5041 cagtttgatt accagaagaa gctggacaac agggagaagg agagggtggc agcaaggcag
     5101 gcctggtctg tggtgggcac aatcaaggat ctgaagcagg gctatctgag ccaggtcatc
     5161 cacgagatcg tggacctgat gatccactac caggccgtgg tggtgctgga gaacctgaat
     5221 ttcggcttta agagcaagag gaccggcatc gccgagaagg ccgtgtacca gcagttcgag
     5281 aagatgctga tcgataagct gaattgcctg gtgctgaagg actatccagc agagaaagtg
     5341 ggaggcgtgc tgaacccata ccagctgaca gaccagttca cctcctttgc caagatgggc
     5401 acccagtctg gcttcctgtt ttacgtgcct gccccatata catctaagat cgatcccctg
     5461 accggcttcg tggacccctt cgtgtggaaa accatcaaga atcacgagag ccgcaagcac
     5521 ttcctggagg gcttcgactt tctgcactac gacgtgaaaa ccggcgactt catcctgcac
     5581 tttaagatga acagaaatct gtccttccag aggggcctgc ccggctttat gcctgcatgg
     5641 gatatcgtgt tcgagaagaa cgagacacag tttgacgcca agggcacccc tttcatcgcc
     5701 ggcaagagaa tcgtgccagt gatcgagaat cacagattca ccggcagata ccgggacctg
     5761 tatcctgcca acgagctgat cgccctgctg gaggagaagg gcatcgtgtt cagggatggc
     5821 tccaacatcc tgccaaagct gctggagaat gacgattctc acgccatcga caccatggtg
     5881 gccctgatcc gcagcgtgct gcagatgcgg aactccaatg ccgccacagg cgaggactat
     5941 atcaacagcc ccgtgcgcga tctgaatggc gtgtgcttcg actcccggtt tcagaaccca
     6001 gagtggccca tggacgccga tgccaatggc gcctaccaca tcgccctgaa gggccagctg
     6061 ctgctgaatc acctgaagga gagcaaggat ctgaagctgc agaacggcat ctccaatcag
     6121 gactggctgg cctacatcca ggagctgcgc aacaaaaggc cggcggccac gaaaaaggcc
     6181 ggccaggcaa aaaagaaaaa gggatcctac ccatacgatg ttccagatta cgcttatccc
     6241 tacgacgtgc ctgattatgc atacccatat gatgtccccg actatgccta agctttgaga
     6301 ccacgaa
//
