LOCUS       sequence-227457-        6309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, AsCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6309)
  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 6309)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2019-02-27"; volume: "14(2):e0211598. doi"; pages: " 
            10.1371/journal.pone.0211598. eCollection 2019"
COMMENT     Sequence Label: pEPOR0SP0008
FEATURES             Location/Qualifiers
     source          1..6309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             436..1227
                     /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      1398..1986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1887..1906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2140..2157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2235..6155
                     /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      5875..5884
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6156..6203
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6210..6290
                     /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 tttttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
      361 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
      421 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
      481 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
      541 tccgcagtgg atggcggcct gaagccacac agcgatattg atttgctggt tacggtgacc
      601 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
      661 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
      721 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
      781 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
      841 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
      901 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
      961 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     1021 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     1081 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     1141 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccatta cgtaaaaggc
     1201 gagatcacca aggtagtcgg caaataactg tcagaccaag tttactcata tatactttag
     1261 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1321 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1381 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1441 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1501 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1561 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1621 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1681 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1741 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1801 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1861 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1921 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1981 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2041 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2101 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2161 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatca
     2221 ctctgtggtc tcaaatgaca cagttcgagg gctttaccaa cctgtatcag gtgagcaaga
     2281 cactgcggtt tgagctgatc ccacagggca agaccctgaa gcacatccag gagcagggct
     2341 tcatcgagga ggacaaggcc cgcaatgatc actacaagga gctgaagccc atcatcgatc
     2401 ggatctacaa gacctatgcc gaccagtgcc tgcagctggt gcagctggat tgggagaacc
     2461 tgagcgccgc catcgactcc tatagaaagg agaaaaccga ggagacaagg aacgccctga
     2521 tcgaggagca ggccacatat cgcaatgcca tccacgacta cttcatcggc cggacagaca
     2581 acctgaccga tgccatcaat aagagacacg ccgagatcta caagggcctg ttcaaggccg
     2641 agctgtttaa tggcaaggtg ctgaagcagc tgggcaccgt gaccacaacc gagcacgaga
     2701 acgccctgct gcggagcttc gacaagttta caacctactt ctccggcttt tatgagaaca
     2761 ggaagaacgt gttcagcgcc gaggatatca gcacagccat cccacaccgc atcgtgcagg
     2821 acaacttccc caagtttaag gagaattgtc acatcttcac acgcctgatc accgccgtgc
     2881 ccagcctgcg ggagcacttt gagaacgtga agaaggccat cggcatcttc gtgagcacct
     2941 ccatcgagga ggtgttttcc ttcccttttt ataaccagct gctgacacag acccagatcg
     3001 acctgtataa ccagctgctg ggaggaatct ctcgggaggc aggcaccgag aagatcaagg
     3061 gcctgaacga ggtgctgaat ctggccatcc agaagaatga tgagacagcc cacatcatcg
     3121 cctccctgcc acacagattc atccccctgt ttaagcagat cctgtccgat aggaacaccc
     3181 tgtctttcat cctggaggag tttaagagcg acgaggaagt gatccagtcc ttctgcaagt
     3241 acaagacact gctgagaaac gagaacgtgc tggagacagc cgaggccctg tttaacgagc
     3301 tgaacagcat cgacctgaca cacatcttca tcagccacaa gaagctggag acaatcagca
     3361 gcgccctgtg cgaccactgg gatacactga ggaatgccct gtatgagcgg agaatctccg
     3421 agctgacagg caagatcacc aagtctgcca aggagaaggt gcagcgcagc ctgaagcacg
     3481 aggatatcaa cctgcaggag atcatctctg ccgcaggcaa ggagctgagc gaggccttca
     3541 agcagaaaac cagcgagatc ctgtcccacg cacacgccgc cctggatcag ccactgccta
     3601 caaccctgaa gaagcaggag gagaaggaga tcctgaagtc tcagctggac agcctgctgg
     3661 gcctgtacca cctgctggac tggtttgccg tggatgagtc caacgaggtg gaccccgagt
     3721 tctctgcccg gctgaccggc atcaagctgg agatggagcc ttctctgagc ttctacaaca
     3781 aggccagaaa ttatgccacc aagaagccct actccgtgga gaagttcaag ctgaactttc
     3841 agatgcctac actggcctct ggctgggacg tgaataagga gaagaacaat ggcgccatcc
     3901 tgtttgtgaa gaacggcctg tactatctgg gcatcatgcc aaagcagaag ggcaggtata
     3961 aggccctgag cttcgagccc acagagaaaa ccagcgaggg ctttgataag atgtactatg
     4021 actacttccc tgatgccgcc aagatgatcc caaagtgcag cacccagctg aaggccgtga
     4081 cagcccactt tcagacccac acaaccccca tcctgctgtc caacaatttc atcgagcctc
     4141 tggagatcac aaaggagatc tacgacctga acaatcctga gaaggagcca aagaagtttc
     4201 agacagccta cgccaagaaa accggcgacc agaagggcta cagagaggcc ctgtgcaagt
     4261 ggatcgactt cacaagggat tttctgtcca agtataccaa gacaacctct atcgatctgt
     4321 ctagcctgcg gccatcctct cagtataagg acctgggcga gtactatgcc gagctgaatc
     4381 ccctgctgta ccacatcagc ttccagagaa tcgccgagaa ggagatcatg gatgccgtgg
     4441 agacaggcaa gctgtacctg ttccagatct ataacaagga ctttgccaag ggccaccacg
     4501 gcaagcctaa tctgcacaca ctgtattgga ccggcctgtt ttctccagag aacctggcca
     4561 agacaagcat caagctgaat ggccaggccg agctgttcta ccgccctaag tccaggatga
     4621 agaggatggc acaccggctg ggagagaaga tgctgaacaa gaagctgaag gatcagaaaa
     4681 ccccaatccc cgacaccctg taccaggagc tgtacgacta tgtgaatcac agactgtccc
     4741 acgacctgtc tgatgaggcc agggccctgc tgcccaacgt gatcaccaag gaggtgtctc
     4801 acgagatcat caaggatagg cgctttacca gcgacaagtt ctttttccac gtgcctatca
     4861 cactgaacta tcaggccgcc aattccccat ctaagttcaa ccagagggtg aatgcctacc
     4921 tgaaggagca ccccgagaca cctatcatcg gcatcgatcg gggcgagaga aacctgatct
     4981 atatcacagt gatcgactcc accggcaaga tcctggagca gcggagcctg aacaccatcc
     5041 agcagtttga ttaccagaag aagctggaca acagggagaa ggagagggtg gcagcaaggc
     5101 aggcctggtc tgtggtgggc acaatcaagg atctgaagca gggctatctg agccaggtca
     5161 tccacgagat cgtggacctg atgatccact accaggccgt ggtggtgctg gagaacctga
     5221 atttcggctt taagagcaag aggaccggca tcgccgagaa ggccgtgtac cagcagttcg
     5281 agaagatgct gatcgataag ctgaattgcc tggtgctgaa ggactatcca gcagagaaag
     5341 tgggaggcgt gctgaaccca taccagctga cagaccagtt cacctccttt gccaagatgg
     5401 gcacccagtc tggcttcctg ttttacgtgc ctgccccata tacatctaag atcgatcccc
     5461 tgaccggctt cgtggacccc ttcgtgtgga aaaccatcaa gaatcacgag agccgcaagc
     5521 acttcctgga gggcttcgac tttctgcact acgacgtgaa aaccggcgac ttcatcctgc
     5581 actttaagat gaacagaaat ctgtccttcc agaggggcct gcccggcttt atgcctgcat
     5641 gggatatcgt gttcgagaag aacgagacac agtttgacgc caagggcacc cctttcatcg
     5701 ccggcaagag aatcgtgcca gtgatcgaga atcacagatt caccggcaga taccgggacc
     5761 tgtatcctgc caacgagctg atcgccctgc tggaggagaa gggcatcgtg ttcagggatg
     5821 gctccaacat cctgccaaag ctgctggaga atgacgattc tcacgccatc gacaccatgg
     5881 tggccctgat ccgcagcgtg ctgcagatgc ggaactccaa tgccgccaca ggcgaggact
     5941 atatcaacag ccccgtgcgc gatctgaatg gcgtgtgctt cgactcccgg tttcagaacc
     6001 cagagtggcc catggacgcc gatgccaatg gcgcctacca catcgccctg aagggccagc
     6061 tgctgctgaa tcacctgaag gagagcaagg atctgaagct gcagaacggc atctccaatc
     6121 aggactggct ggcctacatc caggagctgc gcaacaaaag gccggcggcc acgaaaaagg
     6181 ccggccaggc aaaaaagaaa aagggatcct acccatacga tgttccagat tacgcttatc
     6241 cctacgacgt gcctgattat gcatacccat atgatgtccc cgactatgcc taagctttga
     6301 gaccacgaa
//
