LOCUS       sequence-220552-        8879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter expression plasmid for human codon optimized FnCas12a 
            nuclease with C-terminal NLS and HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8879)
  AUTHORS   Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, 
            Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L, 
            Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas12a variants with increased activities and 
            improved targeting ranges for gene, epigenetic and base editing.
  JOURNAL   Nat Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi: 
            10.1038/s41587-018-0011-0.
  PUBMED    30742127
REFERENCE   2  (bases 1 to 8879)
  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: "Nat 
            Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi: 
            10.1038/s41587-018-0011-0."
COMMENT     Sequence Label: pCAG-hFnCas12a-NLS(nucleoplasmin)-3xHA (AAS1472)
FEATURES             Location/Qualifiers
     source          1..8879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5677..5724
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5731..5811
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(5901..5920)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5966..6021
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6020..6039)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6382..6398)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6382..6398)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6395..6417)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6406..6422
                     /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(6430..6436,6437..6454,6455..6460))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6475..6496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6555..6750
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6601..6736
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6756..6890
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6793..6812)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6847..6866
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6958..6975)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7129..7717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7209..7228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7888..8679,8680..8748))
                     /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     8511..8530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8749..8853)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tctgcagtcg
     1741 acggtaccgc gggcccggga tccaccggtc gccaccatga gcatctacca ggagttcgtc
     1801 aacaagtatt cactgagtaa gacactgcgg ttcgagctga tcccacaggg caagacactg
     1861 gagaacatca aggcccgagg cctgattctg gacgatgaga agcgggcaaa agactataag
     1921 aaagccaagc agatcattga taaataccac cagttcttta tcgaggaaat tctgagctcc
     1981 gtgtgcatca gtgaggatct gctgcagaat tactcagacg tgtacttcaa gctgaagaag
     2041 agcgacgatg acaacctgca gaaggacttc aagtccgcca aggacaccat caagaaacag
     2101 attagcgagt acatcaagga ctccgaaaag tttaaaaatc tgttcaacca gaatctgatc
     2161 gatgctaaga aaggccagga gtccgacctg atcctgtggc tgaaacagtc taaggacaat
     2221 gggattgaac tgttcaaggc taactccgat atcactgata ttgacgaggc actggaaatc
     2281 atcaagagct tcaagggatg gaccacatac tttaaaggct tccacgagaa ccgcaagaac
     2341 gtgtactcca gcaacgacat tcctacctcc atcatctacc gaatcgtcga tgacaatctg
     2401 ccaaagttcc tggagaacaa ggccaaatat gaatctctga aggacaaagc tcccgaggca
     2461 attaattacg aacagatcaa gaaagatctg gctgaggaac tgacattcga tatcgactat
     2521 aagactagcg aggtgaacca gagggtcttt tccctggacg aggtgtttga aatcgccaat
     2581 ttcaacaatt acctgaacca gtccggcatt actaaattca ataccatcat tggcgggaag
     2641 tttgtgaacg gggagaatac caagcgcaag ggaattaacg aatacatcaa tctgtatagc
     2701 cagcagatca acgacaaaac tctgaagaaa tacaagatgt ctgtgctgtt caaacagatc
     2761 ctgagtgata ccgagtccaa gtcttttgtc attgataaac tggaagatga ctcagacgtg
     2821 gtcactacca tgcagagctt ttatgagcag atcgccgctt tcaagacagt ggaggaaaaa
     2881 tctattaagg aaactctgag tctgctgttc gatgacctga aagcccagaa gctggacctg
     2941 agtaagatct acttcaaaaa cgataagagt ctgacagacc tgtcacagca ggtgtttgat
     3001 gactattccg tgattgggac cgccgtcctg gagtacatta cacagcagat cgctccaaag
     3061 aacctggata atccctctaa gaaagagcag gaactgatcg ctaagaaaac cgagaaggca
     3121 aaatatctga gtctggaaac aattaagctg gcactggagg agttcaacaa gcacagggat
     3181 attgacaaac agtgccgctt tgaggaaatc ctggccaact tcgcagccat ccccatgatt
     3241 tttgatgaga tcgcccagaa caaagacaat ctggctcaga tcagtattaa gtaccagaac
     3301 cagggcaaga aagacctgct gcaggcttca gcagaagatg acgtgaaagc catcaaggat
     3361 ctgctggacc agaccaacaa tctgctgcac aagctgaaaa tcttccatat tagtcagtca
     3421 gaggataagg ctaatatcct ggataaagac gaacacttct acctggtgtt cgaggaatgt
     3481 tacttcgagc tggcaaacat tgtccccctg tataacaaga ttaggaacta catcacacag
     3541 aagccttact ctgacgagaa gtttaaactg aacttcgaaa atagtaccct ggccaacggg
     3601 tgggataaga acaaggagcc tgacaacaca gctatcctgt tcatcaagga tgacaagtac
     3661 tatctgggag tgatgaataa gaaaaacaat aagatcttcg atgacaaagc cattaaggag
     3721 aacaaagggg aaggatacaa gaaaatcgtg tataagctgc tgcccggcgc aaataagatg
     3781 ctgcctaagg tgttcttcag cgccaagagt atcaaattct acaacccatc cgaggacatc
     3841 ctgcggatta gaaatcactc aacacatact aagaacggga gcccccagaa gggatatgag
     3901 aaatttgagt tcaacatcga ggattgcagg aagtttattg acttctacaa gcagagcatc
     3961 tccaaacacc ctgaatggaa ggattttggc ttccggtttt ccgacacaca gagatataac
     4021 tctatcgacg agttctaccg cgaggtggaa aatcaggggt ataagctgac ttttgagaac
     4081 atttctgaaa gttacatcga cagcgtggtc aatcagggaa agctgtacct gttccagatc
     4141 tataacaaag atttttcagc atacagcaag ggcagaccaa acctgcatac actgtactgg
     4201 aaggccctgt tcgatgagag gaatctgcag gacgtggtct ataaactgaa cggagaggcc
     4261 gaactgtttt accggaagca gtctattcct aagaaaatca ctcacccagc taaggaggcc
     4321 atcgctaaca agaacaagga caatcctaag aaagagagcg tgttcgaata cgatctgatt
     4381 aaggacaagc ggttcaccga agataagttc tttttccatt gtccaatcac cattaacttc
     4441 aagtcaagcg gcgctaacaa gttcaacgac gagatcaatc tgctgctgaa ggaaaaagca
     4501 aacgatgtgc acatcctgag cattgaccga ggagagcggc atctggccta ctataccctg
     4561 gtggatggca aagggaatat cattaagcag gatacattca acatcattgg caatgaccgg
     4621 atgaaaacca actaccacga taaactggct gcaatcgaga aggatagaga ctcagctagg
     4681 aaggactgga agaaaatcaa caacattaag gagatgaagg aaggctatct gagccaggtg
     4741 gtccatgaga ttgcaaagct ggtcatcgaa tacaatgcca ttgtggtgtt cgaggatctg
     4801 aacttcggct ttaagagggg gcgctttaag gtggaaaaac aggtctatca gaagctggag
     4861 aaaatgctga tcgaaaagct gaattacctg gtgtttaaag ataacgagtt cgacaagacc
     4921 ggaggcgtcc tgagagccta ccagctgaca gctccctttg aaactttcaa gaaaatggga
     4981 aaacagacag gcatcatcta ctatgtgcca gccggattca cttccaagat ctgccccgtg
     5041 accggctttg tcaaccagct gtaccctaaa tatgagtcag tgagcaagtc ccaggaattt
     5101 ttcagcaagt tcgataagat ctgttataat ctggacaagg ggtacttcga gttttccttc
     5161 gattacaaga acttcggcga caaggccgct aaggggaaat ggaccattgc ctccttcgga
     5221 tctcgcctga tcaactttcg aaattccgat aaaaaccaca attgggacac tagggaggtg
     5281 tacccaacca aggagctgga aaagctgctg aaagactact ctatcgagta tggacatggc
     5341 gaatgcatca aggcagccat ctgtggcgag agtgataaga aatttttcgc caagctgacc
     5401 tcagtgctga atacaatcct gcagatgcgg aactcaaaga ccgggacaga actggactat
     5461 ctgattagcc ccgtggctga tgtcaacgga aacttcttcg acagcagaca ggcacccaaa
     5521 aatatgcctc aggatgcaga cgccaacggg gcctaccaca tcgggctgaa gggactgatg
     5581 ctgctgggcc ggatcaagaa caatcaggag gggaagaagc tgaacctggt cattaagaac
     5641 gaggaatact tcgagtttgt ccagaataga aataacaaaa ggccggcggc cacgaaaaag
     5701 gccggccagg caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttat
     5761 ccctacgacg tgcctgatta tgcataccca tatgatgtcc ccgactatgc ctaaagcggc
     5821 cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     5881 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     5941 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     6001 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     6061 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     6121 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     6181 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     6241 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     6301 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     6361 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6421 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6481 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6541 gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     6601 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     6661 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     6721 ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg
     6781 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     6841 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta
     6901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7321 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7561 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     8041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     8101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     8161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     8221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
