LOCUS       sequence-247958-        8139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FnoCas12a-2C-NLS in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8139)
  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 8139)
  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: FnoCas12a-2C-NLS in pCSDest
FEATURES             Location/Qualifiers
     source          1..8139
                     /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             4182..4229
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4236..4316
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             4317..4337
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4373..4507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4397..4416)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4451..4470
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4620..4640)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4620..4638)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4659..4675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4659..4675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4672..4694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4683..4699
                     /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(4707..4713,4714..4731,4732..4737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4752..4773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4890..4907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5061..5649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5141..5160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5820..6611,6612..6680))
                     /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     6443..6462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6681..6785)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6811..7266)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6948..6969)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7160..7179
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(7337..8139,1..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 catgagcatc taccaggagt
      301 tcgtcaacaa gtattcactg agtaagacac tgcggttcga gctgatccca cagggcaaga
      361 cactggagaa catcaaggcc cgaggcctga ttctggacga tgagaagcgg gcaaaagact
      421 ataagaaagc caagcagatc attgataaat accaccagtt ctttatcgag gaaattctga
      481 gctccgtgtg catcagtgag gatctgctgc agaattactc agacgtgtac ttcaagctga
      541 agaagagcga cgatgacaac ctgcagaagg acttcaagtc cgccaaggac accatcaaga
      601 aacagattag cgagtacatc aaggactccg aaaagtttaa aaatctgttc aaccagaatc
      661 tgatcgatgc taagaaaggc caggagtccg acctgatcct gtggctgaaa cagtctaagg
      721 acaatgggat tgaactgttc aaggctaact ccgatatcac tgatattgac gaggcactgg
      781 aaatcatcaa gagcttcaag ggatggacca catactttaa aggcttccac gagaaccgca
      841 agaacgtgta ctccagcaac gacattccta cctccatcat ctaccgaatc gtcgatgaca
      901 atctgccaaa gttcctggag aacaaggcca aatatgaatc tctgaaggac aaagctcccg
      961 aggcaattaa ttacgaacag atcaagaaag atctggctga ggaactgaca ttcgatatcg
     1021 actataagac tagcgaggtg aaccagaggg tcttttccct ggacgaggtg tttgaaatcg
     1081 ccaatttcaa caattacctg aaccagtccg gcattactaa attcaatacc atcattggcg
     1141 ggaagtttgt gaacggggag aataccaagc gcaagggaat taacgaatac atcaatctgt
     1201 atagccagca gatcaacgac aaaactctga agaaatacaa gatgtctgtg ctgttcaaac
     1261 agatcctgag tgataccgag tccaagtctt ttgtcattga taaactggaa gatgactcag
     1321 acgtggtcac taccatgcag agcttttatg agcagatcgc cgctttcaag acagtggagg
     1381 aaaaatctat taaggaaact ctgagtctgc tgttcgatga cctgaaagcc cagaagctgg
     1441 acctgagtaa gatctacttc aaaaacgata agagtctgac agacctgtca cagcaggtgt
     1501 ttgatgacta ttccgtgatt gggaccgccg tcctggagta cattacacag cagatcgctc
     1561 caaagaacct ggataatccc tctaagaaag agcaggaact gatcgctaag aaaaccgaga
     1621 aggcaaaata tctgagtctg gaaacaatta agctggcact ggaggagttc aacaagcaca
     1681 gggatattga caaacagtgc cgctttgagg aaatcctggc caacttcgca gccatcccca
     1741 tgatttttga tgagatcgcc cagaacaaag acaatctggc tcagatcagt attaagtacc
     1801 agaaccaggg caagaaagac ctgctgcagg cttcagcaga agatgacgtg aaagccatca
     1861 aggatctgct ggaccagacc aacaatctgc tgcacaagct gaaaatcttc catattagtc
     1921 agtcagagga taaggctaat atcctggata aagacgaaca cttctacctg gtgttcgagg
     1981 aatgttactt cgagctggca aacattgtcc ccctgtataa caagattagg aactacatca
     2041 cacagaagcc ttactctgac gagaagttta aactgaactt cgaaaatagt accctggcca
     2101 acgggtggga taagaacaag gagcctgaca acacagctat cctgttcatc aaggatgaca
     2161 agtactatct gggagtgatg aataagaaaa acaataagat cttcgatgac aaagccatta
     2221 aggagaacaa aggggaagga tacaagaaaa tcgtgtataa gctgctgccc ggcgcaaata
     2281 agatgctgcc taaggtgttc ttcagcgcca agagtatcaa attctacaac ccatccgagg
     2341 acatcctgcg gattagaaat cactcaacac atactaagaa cgggagcccc cagaagggat
     2401 atgagaaatt tgagttcaac atcgaggatt gcaggaagtt tattgacttc tacaagcaga
     2461 gcatctccaa acaccctgaa tggaaggatt ttggcttccg gttttccgac acacagagat
     2521 ataactctat cgacgagttc taccgcgagg tggaaaatca ggggtataag ctgacttttg
     2581 agaacatttc tgaaagttac atcgacagcg tggtcaatca gggaaagctg tacctgttcc
     2641 agatctataa caaagatttt tcagcataca gcaagggcag accaaacctg catacactgt
     2701 actggaaggc cctgttcgat gagaggaatc tgcaggacgt ggtctataaa ctgaacggag
     2761 aggccgaact gttttaccgg aagcagtcta ttcctaagaa aatcactcac ccagctaagg
     2821 aggccatcgc taacaagaac aaggacaatc ctaagaaaga gagcgtgttc gaatacgatc
     2881 tgattaagga caagcggttc accgaagata agttcttttt ccattgtcca atcaccatta
     2941 acttcaagtc aagcggcgct aacaagttca acgacgagat caatctgctg ctgaaggaaa
     3001 aagcaaacga tgtgcacatc ctgagcattg accgaggaga gcggcatctg gcctactata
     3061 ccctggtgga tggcaaaggg aatatcatta agcaggatac attcaacatc attggcaatg
     3121 accggatgaa aaccaactac cacgataaac tggctgcaat cgagaaggat agagactcag
     3181 ctaggaagga ctggaagaaa atcaacaaca ttaaggagat gaaggaaggc tatctgagcc
     3241 aggtggtcca tgagattgca aagctggtca tcgaatacaa tgccattgtg gtgttcgagg
     3301 atctgaactt cggctttaag agggggcgct ttaaggtgga aaaacaggtc tatcagaagc
     3361 tggagaaaat gctgatcgaa aagctgaatt acctggtgtt taaagataac gagttcgaca
     3421 agaccggagg cgtcctgaga gcctaccagc tgacagctcc ctttgaaact ttcaagaaaa
     3481 tgggaaaaca gacaggcatc atctactatg tgccagccgg attcacttcc aagatctgcc
     3541 ccgtgaccgg ctttgtcaac cagctgtacc ctaaatatga gtcagtgagc aagtcccagg
     3601 aatttttcag caagttcgat aagatctgtt ataatctgga caaggggtac ttcgagtttt
     3661 ccttcgatta caagaacttc ggcgacaagg ccgctaaggg gaaatggacc attgcctcct
     3721 tcggatctcg cctgatcaac tttcgaaatt ccgataaaaa ccacaattgg gacactaggg
     3781 aggtgtaccc aaccaaggag ctggaaaagc tgctgaaaga ctactctatc gagtatggac
     3841 atggcgaatg catcaaggca gccatctgtg gcgagagtga taagaaattt ttcgccaagc
     3901 tgacctcagt gctgaataca atcctgcaga tgcggaactc aaagaccggg acagaactgg
     3961 actatctgat tagccccgtg gctgatgtca acggaaactt cttcgacagc agacaggcac
     4021 ccaaaaatat gcctcaggat gcagacgcca acggggccta ccacatcggg ctgaagggac
     4081 tgatgctgct gggccggatc aagaacaatc aggaggggaa gaagctgaac ctggtcatta
     4141 agaacgagga atacttcgag tttgtccaga atagaaataa caaaaggccg gcggccacga
     4201 aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc atacgatgtt ccagattacg
     4261 cttatcccta cgacgtgcct gattatgcat acccatatga tgtccccgac tatgccccaa
     4321 agaagaagcg gaaggtctaa tgatctagaa ctatagtgag tcgtattacg tagatccaga
     4381 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     4441 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     4501 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     4561 ggttttttaa ttcgcggccg cggcgccaat gcattgggcc cggtacccag cttttgttcc
     4621 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     4681 aattgttatc cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc
     4741 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4801 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4861 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4921 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4981 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5281 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5341 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5401 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5461 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5521 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5581 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5641 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5701 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5761 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5821 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5881 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5941 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6001 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6061 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6121 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6181 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6241 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6301 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6361 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6421 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6481 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6541 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6601 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6661 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6721 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6781 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     6841 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     6901 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     6961 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     7021 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     7081 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     7141 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     7201 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     7261 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     7321 cttcgctatt acgccagtcg accgccaatt caatatggcg tatatggact catgccaatt
     7381 caatatggtg gatctggacc tgtgccaatt caatatggcg tatatggact cgtgccaatt
     7441 caatatggtg gatctggacc ccagccaatt caatatggcg gacttggcac catgccaatt
     7501 caatatggcg gacctggcac tgtgccaact ggggaggggt ctacttggca cggtgccaag
     7561 tttgaggagg ggtcttggcc ctgtgccaag tccgccatat tgaattggca tggtgccaat
     7621 aatggcggcc atattggcta tatgccagga tcaatatata ggcaatatcc aatatggccc
     7681 tatgccaata tggctattgg ccaggttcaa tactatgtat tggccctatg ccatatagta
     7741 ttccatatat gggttttcct attgacgtag atagcccctc ccaatgggcg gtcccatata
     7801 ccatatatgg ggcttcctaa taccgcccat agccactccc ccattgacgt caatggtctc
     7861 tatatatggt ctttcctatt gacgtcatat gggcggtcct attgacgtat atggcgcctc
     7921 ccccattgac gtcaattacg gtaaatggcc cgcctggctc aatgcccatt gacgtcaata
     7981 ggaccaccca ccattgacgt caatgggatg gctcattgcc cattcatatc cgttctcacg
     8041 ccccctattg acgtcaatga cggtaaatgg cccacttggc agtacatcaa tatctattaa
     8101 tagtaacttg gcaagtacat tactattgga agtacgcca
//
