LOCUS       sequence-319707-        6081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human codon optimized coding sequence of Cas12a from Acidominococcus
            sp (AsCas12a)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6081)
  AUTHORS   Bernabe-Orts JM, Casas-Rodrigo I, Minguet EG, Landolfi V, 
            Garcia-Carpintero V, Gianoglio S, Vazquez-Vilar M, Granell A, Orzaez
            D
  TITLE     Assessment of Cas12a-mediated gene editing efficiency in plants.
  JOURNAL   Plant Biotechnol J. 2019 Apr 5. doi: 10.1111/pbi.13113.
  PUBMED    30950179
REFERENCE   2  (bases 1 to 6081)
  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: "Plant 
            Biotechnol J. 2019 Apr 5. doi: 10.1111/pbi.13113."
COMMENT     Sequence Label: pUPD2 AsCas12a (GB1438)
FEATURES             Location/Qualifiers
     source          1..6081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     458..477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     711..728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(752..809)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             complement(857..904)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(905..4825)
                     /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      1176..1185
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4842..4863
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      4866..4909
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(4913..4931)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             5093..5752
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5159..5178)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5773..5867
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(6050..6081,1..557)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
       61 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
      121 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      181 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      241 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      301 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      361 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
      421 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      481 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      541 ggcggagcct gtggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      601 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
      661 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
      721 tgagcgagga agcctgcata acgcgaagta atcttttcgg ttttaaagaa aaagggcagg
      781 gtggtgacac cttgcccttt tttgccggac tgcagcggcc gctactagtc gcgatgagtg
      841 gtctcaaagc tcactacttt ttcttttttg cctggccggc ctttttcgtg gccgccggcc
      901 ttttgttgcg cagctcctgg atgtaggcca gccagtcctg attggagatg ccgttctgca
      961 gcttcagatc cttgctctcc ttcaggtgat tcagcagcag ctggcccttc agggcgatgt
     1021 ggtaggcgcc attggcatcg gcgtccatgg gccactctgg gttctgaaac cgggagtcga
     1081 agcacacgcc attcagatcg cgcacggggc tgttgatata gtcctcgcct gtggcggcat
     1141 tggagttccg catctgcagc acgctgcgga tcagggccac catggtgtcg atggcgtgag
     1201 aatcgtcatt ctccagcagc tttggcagga tgttggagcc atccctgaac acgatgccct
     1261 tctcctccag cagggcgatc agctcgttgg caggatacag gtcccggtat ctgccggtga
     1321 atctgtgatt ctcgatcact ggcacgattc tcttgccggc gatgaaaggg gtgcccttgg
     1381 cgtcaaactg tgtctcgttc ttctcgaaca cgatatccca tgcaggcata aagccgggca
     1441 ggcccctctg gaaggacaga tttctgttca tcttaaagtg caggatgaag tcgccggttt
     1501 tcacgtcgta gtgcagaaag tcgaagccct ccaggaagtg cttgcggctc tcgtgattct
     1561 tgatggtttt ccacacgaag gggtccacga agccggtcag gggatcgatc ttagatgtat
     1621 atggggcagg cacgtaaaac aggaagccag actgggtgcc catcttggca aaggaggtga
     1681 actggtctgt cagctggtat gggttcagca cgcctcccac tttctctgct ggatagtcct
     1741 tcagcaccag gcaattcagc ttatcgatca gcatcttctc gaactgctgg tacacggcct
     1801 tctcggcgat gccggtcctc ttgctcttaa agccgaaatt caggttctcc agcaccacca
     1861 cggcctggta gtggatcatc aggtccacga tctcgtggat gacctggctc agatagccct
     1921 gcttcagatc cttgattgtg cccaccacag accaggcctg ccttgctgcc accctctcct
     1981 tctccctgtt gtccagcttc ttctggtaat caaactgctg gatggtgttc aggctccgct
     2041 gctccaggat cttgccggtg gagtcgatca ctgtgatata gatcaggttt ctctcgcccc
     2101 gatcgatgcc gatgataggt gtctcggggt gctccttcag gtaggcattc accctctggt
     2161 tgaacttaga tggggaattg gcggcctgat agttcagtgt gataggcacg tggaaaaaga
     2221 acttgtcgct ggtaaagcgc ctatccttga tgatctcgtg agacacctcc ttggtgatca
     2281 cgttgggcag cagggccctg gcctcatcag acaggtcgtg ggacagtctg tgattcacat
     2341 agtcgtacag ctcctggtac agggtgtcgg ggattggggt tttctgatcc ttcagcttct
     2401 tgttcagcat cttctctccc agccggtgtg ccatcctctt catcctggac ttagggcggt
     2461 agaacagctc ggcctggcca ttcagcttga tgcttgtctt ggccaggttc tctggagaaa
     2521 acaggccggt ccaatacagt gtgtgcagat taggcttgcc gtggtggccc ttggcaaagt
     2581 ccttgttata gatctggaac aggtacagct tgcctgtctc cacggcatcc atgatctcct
     2641 tctcggcgat tctctggaag ctgatgtggt acagcagggg attcagctcg gcatagtact
     2701 cgcccaggtc cttatactga gaggatggcc gcaggctaga cagatcgata gaggttgtct
     2761 tggtatactt ggacagaaaa tcccttgtga agtcgatcca cttgcacagg gcctctctgt
     2821 agcccttctg gtcgccggtt ttcttggcgt aggctgtctg aaacttcttt ggctccttct
     2881 caggattgtt caggtcgtag atctcctttg tgatctccag aggctcgatg aaattgttgg
     2941 acagcaggat gggggttgtg tgggtctgaa agtgggctgt cacggccttc agctgggtgc
     3001 tgcactttgg gatcatcttg gcggcatcag ggaagtagtc atagtacatc ttatcaaagc
     3061 cctcgctggt tttctctgtg ggctcgaagc tcagggcctt atacctgccc ttctgctttg
     3121 gcatgatgcc cagatagtac aggccgttct tcacaaacag gatggcgcca ttgttcttct
     3181 ccttattcac gtcccagcca gaggccagtg taggcatctg aaagttcagc ttgaacttct
     3241 ccacggagta gggcttcttg gtggcataat ttctggcctt gttgtagaag ctcagagaag
     3301 gctccatctc cagcttgatg ccggtcagcc gggcagagaa ctcggggtcc acctcgttgg
     3361 actcatccac ggcaaaccag tccagcaggt ggtacaggcc cagcaggctg tccagctgag
     3421 acttcaggat ctccttctcc tcctgcttct tcagggttgt aggcagtggc tgatccaggg
     3481 cggcgtgtgc gtgggacagg atctcgctgg ttttctgctt gaaggcctcg ctcagctcct
     3541 tgcctgcggc agagatgatc tcctgcaggt tgatatcctc gtgcttcagg ctgcgctgca
     3601 ccttctcctt ggcagacttg gtgatcttgc ctgtcagctc ggagattctc cgctcataca
     3661 gggcattcct cagtgtatcc cagtggtcgc acagggcgct gctgattgtc tccagcttct
     3721 tgtggctgat gaagatgtgt gtcaggtcga tgctgttcag ctcgttaaac agggcctcgg
     3781 ctgtctccag cacgttctcg tttctcagca gtgtcttgta cttgcagaag gactggatca
     3841 cttcctcgtc gctcttaaac tcctccagga tgaaagacag ggtgttccta tcggacagga
     3901 tctgcttaaa cagggggatg aatctgtgtg gcagggaggc gatgatgtgg gctgtctcat
     3961 cattcttctg gatggccaga ttcagcacct cgttcaggcc cttgatcttc tcggtgcctg
     4021 cctcccgaga gattcctccc agcagctggt tatacaggtc gatctgggtc tgtgtcagca
     4081 gctggttata aaaagggaag gaaaacacct cctcgatgga ggtgctcacg aagatgccga
     4141 tggccttctt cacgttctca aagtgctccc gcaggctggg cacggcggtg atcaggcgtg
     4201 tgaagatgtg acaattctcc ttaaacttgg ggaagttgtc ctgcacgatg cggtgtggga
     4261 tggctgtgct gatatcctcg gcgctgaaca cgttcttcct gttctcataa aagccggaga
     4321 agtaggttgt aaacttgtcg aagctccgca gcagggcgtt ctcgtgctcg gttgtggtca
     4381 cggtgcccag ctgcttcagc accttgccat taaacagctc ggccttgaac aggcccttgt
     4441 agatctcggc gtgtctctta ttgatggcat cggtcaggtt gtctgtccgg ccgatgaagt
     4501 agtcgtggat ggcattgcga tatgtggcct gctcctcgat cagggcgttc cttgtctcct
     4561 cggttttctc ctttctatag gagtcgatgg cggcgctcag gttctcccaa tccagctgca
     4621 ccagctgcag gcactggtcg gcataggtct tgtagatccg atcgatgatg ggcttcagct
     4681 ccttgtagtg atcattgcgg gccttgtcct cctcgatgaa gccctgctcc tggatgtgct
     4741 tcagggtctt gccctgtggg atcagctcaa accgcagtgt cttgctcacc tgatacaggt
     4801 tggtaaagcc ctcgaactgt gtcattcgag accactcatc gctctagaag cggccgcgaa
     4861 ttccagaaat catccttagc gaaagctaag gatttttttt atctgaaatt ctgcctcgtg
     4921 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4981 actcgagttg atcgggcacg taagaggttc caactttcac cataatgaaa taagatcact
     5041 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa
     5101 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
     5161 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
     5221 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
     5281 tgcccgcctg atgaatgctc atccggaatt tcgtatggca atgaaagacg gtgagctggt
     5341 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
     5401 atcactctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
     5461 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
     5521 tttcgtatca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
     5581 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     5641 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag
     5701 aatgcttaat gaattacaac agtactgcga cgagtggcag ggcggggcgt aatttgatat
     5761 cgagctcgct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     5821 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca agcctcgagc
     5881 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5941 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6001 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6061 tttttctgcg cgtaatctgc t
//
