LOCUS       sequence-247961-        8160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AsCas12a-2C-NLS in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8160)
  AUTHORS   Liu P, Luk K, Shin M, Idrizi F, Kwok S, Roscoe B, Mintzer E, Suresh 
            S, Morrison K, Frazao JB, Bolukbasi MF, Ponnienselvan K, Luban J, 
            Zhu LJ, Lawson ND, Wolfe SA
  TITLE     Enhanced Cas12a editing in mammalian cells and zebrafish.
  JOURNAL   Nucleic Acids Res. 2019 Mar 20. pii: 5403491. doi: 
            10.1093/nar/gkz184.
  PUBMED    30892626
REFERENCE   2  (bases 1 to 8160)
  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: "Nucleic 
            Acids Res. 2019 Mar 20. pii: 5403491. doi: 10.1093/nar/gkz184."
COMMENT     Sequence Label: AsCas12a-2C-NLS in pCSDest
FEATURES             Location/Qualifiers
     source          1..8160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        217..235
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     217..234
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             282..4202
                     /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      3922..3931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4203..4250
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4257..4337
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             4338..4358
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4394..4528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4418..4437)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4472..4491
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4641..4661)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4641..4659)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4680..4696)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4680..4696)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4693..4715)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4704..4720
                     /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(join(4728..4734,4735..4752,4753..4758))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4773..4794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4911..4928)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5082..5670)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5162..5181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5841..6632,6633..6701))
                     /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"
     primer_bind     6464..6483
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6702..6806)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6832..7287)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6969..6990)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7181..7200
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(7358..8160,1..182)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc
       61 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga
      121 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga
      181 accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca
      241 agctacttgt tctttttgca ggatcccatc gattcgaatt catgacacag ttcgagggct
      301 ttaccaacct gtatcaggtg agcaagacac tgcggtttga gctgatccca cagggcaaga
      361 ccctgaagca catccaggag cagggcttca tcgaggagga caaggcccgc aatgatcact
      421 acaaggagct gaagcccatc atcgatcgga tctacaagac ctatgccgac cagtgcctgc
      481 agctggtgca gctggattgg gagaacctga gcgccgccat cgactcctat agaaaggaga
      541 aaaccgagga gacaaggaac gccctgatcg aggagcaggc cacatatcgc aatgccatcc
      601 acgactactt catcggccgg acagacaacc tgaccgatgc catcaataag agacacgccg
      661 agatctacaa gggcctgttc aaggccgagc tgtttaatgg caaggtgctg aagcagctgg
      721 gcaccgtgac cacaaccgag cacgagaacg ccctgctgcg gagcttcgac aagtttacaa
      781 cctacttctc cggcttttat gagaacagga agaacgtgtt cagcgccgag gatatcagca
      841 cagccatccc acaccgcatc gtgcaggaca acttccccaa gtttaaggag aattgtcaca
      901 tcttcacacg cctgatcacc gccgtgccca gcctgcggga gcactttgag aacgtgaaga
      961 aggccatcgg catcttcgtg agcacctcca tcgaggaggt gttttccttc cctttttata
     1021 accagctgct gacacagacc cagatcgacc tgtataacca gctgctggga ggaatctctc
     1081 gggaggcagg caccgagaag atcaagggcc tgaacgaggt gctgaatctg gccatccaga
     1141 agaatgatga gacagcccac atcatcgcct ccctgccaca cagattcatc cccctgttta
     1201 agcagatcct gtccgatagg aacaccctgt ctttcatcct ggaggagttt aagagcgacg
     1261 aggaagtgat ccagtccttc tgcaagtaca agacactgct gagaaacgag aacgtgctgg
     1321 agacagccga ggccctgttt aacgagctga acagcatcga cctgacacac atcttcatca
     1381 gccacaagaa gctggagaca atcagcagcg ccctgtgcga ccactgggat acactgagga
     1441 atgccctgta tgagcggaga atctccgagc tgacaggcaa gatcaccaag tctgccaagg
     1501 agaaggtgca gcgcagcctg aagcacgagg atatcaacct gcaggagatc atctctgccg
     1561 caggcaagga gctgagcgag gccttcaagc agaaaaccag cgagatcctg tcccacgcac
     1621 acgccgccct ggatcagcca ctgcctacaa ccctgaagaa gcaggaggag aaggagatcc
     1681 tgaagtctca gctggacagc ctgctgggcc tgtaccacct gctggactgg tttgccgtgg
     1741 atgagtccaa cgaggtggac cccgagttct ctgcccggct gaccggcatc aagctggaga
     1801 tggagccttc tctgagcttc tacaacaagg ccagaaatta tgccaccaag aagccctact
     1861 ccgtggagaa gttcaagctg aactttcaga tgcctacact ggcctctggc tgggacgtga
     1921 ataaggagaa gaacaatggc gccatcctgt ttgtgaagaa cggcctgtac tatctgggca
     1981 tcatgccaaa gcagaagggc aggtataagg ccctgagctt cgagcccaca gagaaaacca
     2041 gcgagggctt tgataagatg tactatgact acttccctga tgccgccaag atgatcccaa
     2101 agtgcagcac ccagctgaag gccgtgacag cccactttca gacccacaca acccccatcc
     2161 tgctgtccaa caatttcatc gagcctctgg agatcacaaa ggagatctac gacctgaaca
     2221 atcctgagaa ggagccaaag aagtttcaga cagcctacgc caagaaaacc ggcgaccaga
     2281 agggctacag agaggccctg tgcaagtgga tcgacttcac aagggatttt ctgtccaagt
     2341 ataccaagac aacctctatc gatctgtcta gcctgcggcc atcctctcag tataaggacc
     2401 tgggcgagta ctatgccgag ctgaatcccc tgctgtacca catcagcttc cagagaatcg
     2461 ccgagaagga gatcatggat gccgtggaga caggcaagct gtacctgttc cagatctata
     2521 acaaggactt tgccaagggc caccacggca agcctaatct gcacacactg tattggaccg
     2581 gcctgttttc tccagagaac ctggccaaga caagcatcaa gctgaatggc caggccgagc
     2641 tgttctaccg ccctaagtcc aggatgaaga ggatggcaca ccggctggga gagaagatgc
     2701 tgaacaagaa gctgaaggat cagaaaaccc caatccccga caccctgtac caggagctgt
     2761 acgactatgt gaatcacaga ctgtcccacg acctgtctga tgaggccagg gccctgctgc
     2821 ccaacgtgat caccaaggag gtgtctcacg agatcatcaa ggataggcgc tttaccagcg
     2881 acaagttctt tttccacgtg cctatcacac tgaactatca ggccgccaat tccccatcta
     2941 agttcaacca gagggtgaat gcctacctga aggagcaccc cgagacacct atcatcggca
     3001 tcgatcgggg cgagagaaac ctgatctata tcacagtgat cgactccacc ggcaagatcc
     3061 tggagcagcg gagcctgaac accatccagc agtttgatta ccagaagaag ctggacaaca
     3121 gggagaagga gagggtggca gcaaggcagg cctggtctgt ggtgggcaca atcaaggatc
     3181 tgaagcaggg ctatctgagc caggtcatcc acgagatcgt ggacctgatg atccactacc
     3241 aggccgtggt ggtgctggag aacctgaatt tcggctttaa gagcaagagg accggcatcg
     3301 ccgagaaggc cgtgtaccag cagttcgaga agatgctgat cgataagctg aattgcctgg
     3361 tgctgaagga ctatccagca gagaaagtgg gaggcgtgct gaacccatac cagctgacag
     3421 accagttcac ctcctttgcc aagatgggca cccagtctgg cttcctgttt tacgtgcctg
     3481 ccccatatac atctaagatc gatcccctga ccggcttcgt ggaccccttc gtgtggaaaa
     3541 ccatcaagaa tcacgagagc cgcaagcact tcctggaggg cttcgacttt ctgcactacg
     3601 acgtgaaaac cggcgacttc atcctgcact ttaagatgaa cagaaatctg tccttccaga
     3661 ggggcctgcc cggctttatg cctgcatggg atatcgtgtt cgagaagaac gagacacagt
     3721 ttgacgccaa gggcacccct ttcatcgccg gcaagagaat cgtgccagtg atcgagaatc
     3781 acagattcac cggcagatac cgggacctgt atcctgccaa cgagctgatc gccctgctgg
     3841 aggagaaggg catcgtgttc agggatggct ccaacatcct gccaaagctg ctggagaatg
     3901 acgattctca cgccatcgac accatggtgg ccctgatccg cagcgtgctg cagatgcgga
     3961 actccaatgc cgccacaggc gaggactata tcaacagccc cgtgcgcgat ctgaatggcg
     4021 tgtgcttcga ctcccggttt cagaacccag agtggcccat ggacgccgat gccaatggcg
     4081 cctaccacat cgccctgaag ggccagctgc tgctgaatca cctgaaggag agcaaggatc
     4141 tgaagctgca gaacggcatc tccaatcagg actggctggc ctacatccag gagctgcgca
     4201 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag ggatcctacc
     4261 catacgatgt tccagattac gcttatccct acgacgtgcc tgattatgca tacccatatg
     4321 atgtccccga ctatgcccca aagaagaagc ggaaggtcta atgatctaga actatagtga
     4381 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
     4441 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4501 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4561 ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc
     4621 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     4681 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4741 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4801 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4861 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4921 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4981 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5041 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5101 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5161 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5221 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5281 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5341 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5401 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5461 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5521 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5581 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5641 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5701 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5761 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5821 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5881 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5941 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6001 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6061 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6121 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6181 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6241 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6301 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6361 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6421 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6481 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6541 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6601 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6661 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6721 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6781 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     6841 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6901 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6961 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     7021 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     7081 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     7141 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     7201 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     7261 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     7321 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc
     7381 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     7441 gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc
     7501 ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac tggggagggg
     7561 tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata
     7621 ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg atcaatatat
     7681 aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca atactatgta
     7741 ttggccctat gccatatagt attccatata tgggttttcc tattgacgta gatagcccct
     7801 cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc
     7861 cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc
     7921 tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct
     7981 caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat ggctcattgc
     8041 ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg gcccacttgg
     8101 cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg aagtacgcca
//
