LOCUS       sequence-215569-        9088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter expression plasmid for human codon optimized MbCas12a 
            nuclease with C-terminal NLS and HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9088)
  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 9088)
  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-hMbCas12a-NLS(nucleoplasmin)-3xHA (AAS2134)
FEATURES             Location/Qualifiers
     source          1..9088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5888..5935
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5942..6022
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6112..6131)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6177..6232
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6231..6250)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6591..6607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6591..6607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6604..6626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6615..6631
                     /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(6639..6645,6646..6663,6664..6669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6684..6705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6764..6959
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6810..6945
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6965..7099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7002..7021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7056..7075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7167..7184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7338..7926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7418..7437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8097..8888,8889..8957))
                     /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     8720..8739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8958..9062)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc
     1741 gcgggcccgg gatccaccgg tcgccaccat gctgttccag gactttaccc acctgtatcc
     1801 actgtccaag acagtgagat ttgagctgaa gcccatcgat aggaccctgg agcacatcca
     1861 cgccaagaac ttcctgtctc aggacgagac aatggccgat atgcaccaga aggtgaaagt
     1921 gatcctggac gattaccacc gcgacttcat cgccgatatg atgggcgagg tgaagctgac
     1981 caagctggcc gagttctatg acgtgtacct gaagtttcgg aagaacccaa aggacgatga
     2041 gctgcagaag cagctgaagg atctgcaggc cgtgctgaga aaggagatcg tgaagcccat
     2101 cggcaatggc ggcaagtata aggccggcta cgacaggctg ttcggcgcca agctgtttaa
     2161 ggacggcaag gagctgggcg atctggccaa gttcgtgatc gcacaggagg gagagagctc
     2221 cccaaagctg gcccacctgg cccacttcga gaagttttcc acctatttca caggctttca
     2281 cgataaccgg aagaatatgt attctgacga ggataagcac accgccatcg cctaccgcct
     2341 gatccacgag aacctgcccc ggtttatcga caatctgcag atcctgacca caatcaagca
     2401 gaagcactct gccctgtacg atcagatcat caacgagctg accgccagcg gcctggacgt
     2461 gtctctggcc agccacctgg atggctatca caagctgctg acacaggagg gcatcaccgc
     2521 ctacaataca ctgctgggag gaatctccgg agaggcaggc tctcctaaga tccagggcat
     2581 caacgagctg atcaattctc accacaacca gcactgccac aagagcgaga gaatcgccaa
     2641 gctgaggcca ctgcacaagc agatcctgtc cgacggcatg agcgtgtcct tcctgccctc
     2701 taagtttgcc gacgatagcg agatgtgcca ggccgtgaac gagttctatc gccactacgc
     2761 cgacgtgttc gccaaggtgc agagcctgtt cgacggcttt gacgatcacc agaaggatgg
     2821 catctacgtg gagcacaaga acctgaatga gctgtccaag caggccttcg gcgactttgc
     2881 actgctggga cgcgtgctgg acggatacta tgtggatgtg gtgaatccag agttcaacga
     2941 gcggtttgcc aaggccaaga ccgacaatgc caaggccaag ctgacaaagg agaaggataa
     3001 gttcatcaag ggcgtgcact ccctggcctc tctggagcag gccatcgagc actataccgc
     3061 aaggcacgac gatgagagcg tgcaggcagg caagctggga cagtacttca agcacggcct
     3121 ggccggagtg gacaacccca tccagaagat ccacaacaat cacagcacca tcaagggctt
     3181 tctggagagg gagcgccctg caggagagag agccctgcca aagatcaagt ccggcaagaa
     3241 tcctgagatg acacagctga ggcagctgaa ggagctgctg gataacgccc tgaatgtggc
     3301 ccacttcgcc aagctgctga ccacaaagac cacactggac aatcaggatg gcaacttcta
     3361 tggcgagttt ggcgtgctgt acgacgagct ggccaagatc cccaccctgt ataacaaggt
     3421 gagagattac ctgagccaga agcctttctc caccgagaag tacaagctga actttggcaa
     3481 tccaacactg ctgaatggct gggacctgaa caaggagaag gataatttcg gcgtgatcct
     3541 gcagaaggac ggctgctact atctggccct gctggacaag gcccacaaga aggtgtttga
     3601 taacgcccct aatacaggca agagcatcta tcagaagatg atctataagt acctggaggt
     3661 gaggaagcag ttccccaagg tgttcttttc caaggaggcc atcgccatca actaccaccc
     3721 ttctaaggag ctggtggaga tcaaggacaa gggccggcag agatccgacg atgagcgcct
     3781 gaagctgtat cggtttatcc tggagtgtct gaagatccac cctaagtacg ataagaagtt
     3841 cgagggcgcc atcggcgaca tccagctgtt taagaaggat aagaagggca gagaggtgcc
     3901 aatcagcgag aaggacctgt tcgataagat caacggcatc ttttctagca agcctaagct
     3961 ggagatggag gacttcttta tcggcgagtt caagaggtat aacccaagcc aggacctggt
     4021 ggatcagtat aatatctaca agaagatcga ctccaacgat aatcgcaaga aggagaattt
     4081 ctacaacaat caccccaagt ttaagaagga tctggtgcgg tactattacg agtctatgtg
     4141 caagcacgag gagtgggagg agagcttcga gttttccaag aagctgcagg acatcggctg
     4201 ttacgtggat gtgaacgagc tgtttaccga gatcgagaca cggagactga attataagat
     4261 ctccttctgc aacatcaatg ccgactacat cgatgagctg gtggagcagg gccagctgta
     4321 tctgttccag atctacaaca aggacttttc cccaaaggcc cacggcaagc ccaatctgca
     4381 caccctgtac ttcaaggccc tgttttctga ggacaacctg gccgatccta tctataagct
     4441 gaatggcgag gcccagatct tctacagaaa ggcctccctg gacatgaacg agacaacaat
     4501 ccacagggcc ggcgaggtgc tggagaacaa gaatcccgat aatcctaaga agagacagtt
     4561 cgtgtacgac atcatcaagg ataagaggta cacacaggac aagttcatgc tgcacgtgcc
     4621 aatcaccatg aactttggcg tgcagggcat gacaatcaag gagttcaata agaaggtgaa
     4681 ccagtctatc cagcagtatg acgaggtgaa cgtgatcggc atcgatcggg gcgagagaca
     4741 cctgctgtac ctgaccgtga tcaatagcaa gggcgagatc ctggagcagt gttccctgaa
     4801 cgacatcacc acagcctctg ccaatggcac acagatgacc acaccttacc acaagatcct
     4861 ggataagagg gagatcgagc gcctgaacgc ccgggtggga tggggcgaga tcgagacaat
     4921 caaggagctg aagtctggct atctgagcca cgtggtgcac cagatcagcc agctgatgct
     4981 gaagtacaac gccatcgtgg tgctggagga cctgaatttc ggctttaaga ggggccgctt
     5041 taaggtggag aagcagatct atcagaactt cgagaatgcc ctgatcaaga agctgaacca
     5101 cctggtgctg aaggacaagg ccgacgatga gatcggctct tacaagaatg ccctgcagct
     5161 gaccaacaat ttcacagatc tgaagagcat cggcaagcag accggcttcc tgttttatgt
     5221 gcccgcctgg aacacctcta agatcgaccc tgagacaggc tttgtggatc tgctgaagcc
     5281 aagatacgag aacatcgccc agagccaggc cttctttggc aagttcgaca agatctgcta
     5341 taatgccgac aaggattact tcgagtttca catcgactac gccaagttta ccgataaggc
     5401 caagaatagc cgccagatct ggacaatctg ttcccacggc gacaagcggt acgtgtacga
     5461 taagacagcc aaccagaata agggcgccgc caagggcatc aacgtgaatg atgagctgaa
     5521 gtccctgttc gcccgccacc acatcaacga gaagcagccc aacctggtca tggacatctg
     5581 ccagaacaat gataaggagt ttcacaagtc tctgatgtac ctgctgaaaa ccctgctggc
     5641 cctgcggtac agcaacgcct cctctgacga ggatttcatc ctgtcccccg tggcaaacga
     5701 cgagggcgtg ttctttaata gcgccctggc cgacgataca cagcctcaga atgccgatgc
     5761 caacggcgcc taccacatcg ccctgaaggg cctgtggctg ctgaatgagc tgaagaactc
     5821 cgacgatctg aacaaggtga agctggccat cgacaatcag acctggctga atttcgccca
     5881 gaacaggaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc
     5941 ctacccatac gatgttccag attacgctta tccctacgac gtgcctgatt atgcataccc
     6001 atatgatgtc cccgactatg cctaaagcgg ccgcactcct caggtgcagg ctgcctatca
     6061 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     6121 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     6181 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     6241 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     6301 aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt catcagtata
     6361 tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga
     6421 ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac
     6481 atgttttact agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc
     6541 ttctcttatg aagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg
     6601 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6661 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6721 ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag
     6781 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     6841 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     6901 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     6961 agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     7021 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     7081 atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     7141 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     7201 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     7261 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     7321 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     7381 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     7441 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     7501 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     7561 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     7621 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     7681 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7741 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     7801 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7861 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7921 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     7981 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     8041 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     8101 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     8161 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     8221 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     8281 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     8341 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     8401 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     8461 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     8521 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     8581 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     8641 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     8701 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     8761 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8821 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8881 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     8941 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     9001 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     9061 cgcacatttc cccgaaaagt gccacctg
//
