LOCUS       sequence-247967-        7923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LbCas12a-2C-NLS in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7923)
  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 7923)
  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: LbCas12a-2C-NLS in pCSDest
FEATURES             Location/Qualifiers
     source          1..7923
                     /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..3965
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VKH"
     CDS             3966..4013
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4020..4100
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             4101..4121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4157..4291
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4181..4200)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4235..4254
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4404..4424)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4404..4422)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4443..4459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4443..4459)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4456..4478)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4467..4483
                     /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(4491..4497,4498..4515,4516..4521))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4536..4557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4674..4691)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4845..5433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4925..4944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5604..6395,6396..6464))
                     /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     6227..6246
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6465..6569)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6595..7050)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6732..6753)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6944..6963
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(7121..7923,1..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 catgagcaag ctggagaagt
      301 ttacaaactg ctactccctg tctaagaccc tgaggttcaa ggccatccct gtgggcaaga
      361 cccaggagaa catcgacaat aagcggctgc tggtggagga cgagaagaga gccgaggatt
      421 ataagggcgt gaagaagctg ctggatcgct actatctgtc ttttatcaac gacgtgctgc
      481 acagcatcaa gctgaagaat ctgaacaatt acatcagcct gttccggaag aaaaccagaa
      541 ccgagaagga gaataaggag ctggagaacc tggagatcaa tctgcggaag gagatcgcca
      601 aggccttcaa gggcaacgag ggctacaagt ccctgtttaa gaaggatatc atcgagacaa
      661 tcctgccaga gttcctggac gataaggacg agatcgccct ggtgaacagc ttcaatggct
      721 ttaccacagc cttcaccggc ttctttgata acagagagaa tatgttttcc gaggaggcca
      781 agagcacatc catcgccttc aggtgtatca acgagaatct gacccgctac atctctaata
      841 tggacatctt cgagaaggtg gacgccatct ttgataagca cgaggtgcag gagatcaagg
      901 agaagatcct gaacagcgac tatgatgtgg aggatttctt tgagggcgag ttctttaact
      961 ttgtgctgac acaggagggc atcgacgtgt ataacgccat catcggcggc ttcgtgaccg
     1021 agagcggcga gaagatcaag ggcctgaacg agtacatcaa cctgtataat cagaaaacca
     1081 agcagaagct gcctaagttt aagccactgt ataagcaggt gctgagcgat cgggagtctc
     1141 tgagcttcta cggcgagggc tatacatccg atgaggaggt gctggaggtg tttagaaaca
     1201 ccctgaacaa gaacagcgag atcttcagct ccatcaagaa gctggagaag ctgttcaaga
     1261 attttgacga gtactctagc gccggcatct ttgtgaagaa cggccccgcc atcagcacaa
     1321 tctccaagga tatcttcggc gagtggaacg tgatccggga caagtggaat gccgagtatg
     1381 acgatatcca cctgaagaag aaggccgtgg tgaccgagaa gtacgaggac gatcggagaa
     1441 agtccttcaa gaagatcggc tccttttctc tggagcagct gcaggagtac gccgacgccg
     1501 atctgtctgt ggtggagaag ctgaaggaga tcatcatcca gaaggtggat gagatctaca
     1561 aggtgtatgg ctcctctgag aagctgttcg acgccgattt tgtgctggag aagagcctga
     1621 agaagaacga cgccgtggtg gccatcatga aggacctgct ggattctgtg aagagcttcg
     1681 agaattacat caaggccttc tttggcgagg gcaaggagac aaacagggac gagtccttct
     1741 atggcgattt tgtgctggcc tacgacatcc tgctgaaggt ggaccacatc tacgatgcca
     1801 tccgcaatta tgtgacccag aagccctact ctaaggataa gttcaagctg tattttcaga
     1861 accctcagtt catgggcggc tgggacaagg ataaggagac agactatcgg gccaccatcc
     1921 tgagatacgg ctccaagtac tatctggcca tcatggataa gaagtacgcc aagtgcctgc
     1981 agaagatcga caaggacgat gtgaacggca attacgagaa gatcaactat aagctgctgc
     2041 ccggccctaa taagatgctg ccaaaggtgt tcttttctaa gaagtggatg gcctactata
     2101 accccagcga ggacatccag aagatctaca agaatggcac attcaagaag ggcgatatgt
     2161 ttaacctgaa tgactgtcac aagctgatcg acttctttaa ggatagcatc tcccggtatc
     2221 caaagtggtc caatgcctac gatttcaact tttctgagac agagaagtat aaggacatcg
     2281 ccggctttta cagagaggtg gaggagcagg gctataaggt gagcttcgag tctgccagca
     2341 agaaggaggt ggataagctg gtggaggagg gcaagctgta tatgttccag atctataaca
     2401 aggacttttc cgataagtct cacggcacac ccaatctgca caccatgtac ttcaagctgc
     2461 tgtttgacga gaacaatcac ggacagatca ggctgagcgg aggagcagag ctgttcatga
     2521 ggcgcgcctc cctgaagaag gaggagctgg tggtgcaccc agccaactcc cctatcgcca
     2581 acaagaatcc agataatccc aagaaaacca caaccctgtc ctacgacgtg tataaggata
     2641 agaggttttc tgaggaccag tacgagctgc acatcccaat cgccatcaat aagtgcccca
     2701 agaacatctt caagatcaat acagaggtgc gcgtgctgct gaagcacgac gataacccct
     2761 atgtgatcgg catcgatagg ggcgagcgca atctgctgta tatcgtggtg gtggacggca
     2821 agggcaacat cgtggagcag tattccctga acgagatcat caacaacttc aacggcatca
     2881 ggatcaagac agattaccac tctctgctgg acaagaagga gaaggagagg ttcgaggccc
     2941 gccagaactg gacctccatc gagaatatca aggagctgaa ggccggctat atctctcagg
     3001 tggtgcacaa gatctgcgag ctggtggaga agtacgatgc cgtgatcgcc ctggaggacc
     3061 tgaactctgg ctttaagaat agccgcgtga aggtggagaa gcaggtgtat cagaagttcg
     3121 agaagatgct gatcgataag ctgaactaca tggtggacaa gaagtctaat ccttgtgcaa
     3181 caggcggcgc cctgaagggc tatcagatca ccaataagtt cgagagcttt aagtccatgt
     3241 ctacccagaa cggcttcatc ttttacatcc ctgcctggct gacatccaag atcgatccat
     3301 ctaccggctt tgtgaacctg ctgaaaacca agtataccag catcgccgat tccaagaagt
     3361 tcatcagctc ctttgacagg atcatgtacg tgcccgagga ggatctgttc gagtttgccc
     3421 tggactataa gaacttctct cgcacagacg ccgattacat caagaagtgg aagctgtact
     3481 cctacggcaa ccggatcaga atcttccgga atcctaagaa gaacaacgtg ttcgactggg
     3541 aggaggtgtg cctgaccagc gcctataagg agctgttcaa caagtacggc atcaattatc
     3601 agcagggcga tatcagagcc ctgctgtgcg agcagtccga caaggccttc tactctagct
     3661 ttatggccct gatgagcctg atgctgcaga tgcggaacag catcacaggc cgcaccgacg
     3721 tggattttct gatcagccct gtgaagaact ccgacggcat cttctacgat agccggaact
     3781 atgaggccca ggagaatgcc atcctgccaa agaacgccga cgccaatggc gcctataaca
     3841 tcgccagaaa ggtgctgtgg gccatcggcc agttcaagaa ggccgaggac gagaagctgg
     3901 ataaggtgaa gatcgccatc tctaacaagg agtggctgga gtacgcccag accagcgtga
     3961 agcacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatcct
     4021 acccatacga tgttccagat tacgcttatc cctacgacgt gcctgattat gcatacccat
     4081 atgatgtccc cgactatgcc ccaaagaaga agcggaaggt ctaatgatct agaactatag
     4141 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     4201 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4261 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4321 tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc caatgcattg
     4381 ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     4441 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4501 gccgggagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4561 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4621 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4681 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4741 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4801 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4861 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4921 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4981 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5041 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5101 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5161 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5221 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5281 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5341 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5401 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5461 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5521 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5581 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5641 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5701 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5761 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5821 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5881 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5941 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6001 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6061 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6121 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6181 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6241 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6301 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6361 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6421 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6481 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6541 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     6601 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     6661 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     6721 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     6781 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     6841 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     6901 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     6961 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     7021 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     7081 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc aattcaatat
     7141 ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc aattcaatat
     7201 ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc aattcaatat
     7261 ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc aactggggag
     7321 gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc caagtccgcc
     7381 atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc aggatcaata
     7441 tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt tcaatactat
     7501 gtattggccc tatgccatat agtattccat atatgggttt tcctattgac gtagatagcc
     7561 cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc ccatagccac
     7621 tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc atatgggcgg
     7681 tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat ggcccgcctg
     7741 gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg gatggctcat
     7801 tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa atggcccact
     7861 tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat tggaagtacg
     7921 cca
//
