LOCUS       sequence-230476-        9098 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 9098)
  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 9098)
  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..9098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          658..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5892..5939
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5946..6026
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6116..6135)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6181..6236
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6235..6254)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6597..6613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6597..6613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6610..6632)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6621..6637
                     /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(6645..6651,6652..6669,6670..6675))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6690..6711
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6770..6965
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6816..6951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6971..7105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7008..7027)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7062..7081
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7173..7190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7344..7932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7424..7443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8103..8894,8895..8963))
                     /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     8726..8745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8964..9068)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggcgcgccr ggsggggsgg ggsggggsgr ggggsggggs
      541 ggggsgrggc ggagaggkgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      601 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      661 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      841 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattctg cagtcgacgg
     1741 taccgcgggc ccgggatcca ccggtcgcca ccatgctgtt ccaggacttt acccacctgt
     1801 atccactgtc caagacagtg agatttgagc tgaagcccat cgataggacc ctggagcaca
     1861 tccacgccaa gaacttcctg tctcaggacg agacaatggc cgatatgcac cagaaggtga
     1921 aagtgatcct ggacgattac caccgcgact tcatcgccga tatgatgggc gaggtgaagc
     1981 tgaccaagct ggccgagttc tatgacgtgt acctgaagtt tcggaagaac ccaaaggacg
     2041 atgagctgca gaagcagctg aaggatctgc aggccgtgct gagaaaggag atcgtgaagc
     2101 ccatcggcaa tggcggcaag tataaggccg gctacgacag gctgttcggc gccaagctgt
     2161 ttaaggacgg caaggagctg ggcgatctgg ccaagttcgt gatcgcacag gagggagaga
     2221 gctccccaaa gctggcccac ctggcccact tcgagaagtt ttccacctat ttcacaggct
     2281 ttcacgataa ccggaagaat atgtattctg acgaggataa gcacaccgcc atcgcctacc
     2341 gcctgatcca cgagaacctg ccccggttta tcgacaatct gcagatcctg accacaatca
     2401 agcagaagca ctctgccctg tacgatcaga tcatcaacga gctgaccgcc agcggcctgg
     2461 acgtgtctct ggccagccac ctggatggct atcacaagct gctgacacag gagggcatca
     2521 ccgcctacaa tacactgctg ggaggaatct ccggagaggc aggctctcct aagatccagg
     2581 gcatcaacga gctgatcaat tctcaccaca accagcactg ccacaagagc gagagaatcg
     2641 ccaagctgag gccactgcac aagcagatcc tgtccgacgg catgagcgtg tccttcctgc
     2701 cctctaagtt tgccgacgat agcgagatgt gccaggccgt gaacgagttc tatcgccact
     2761 acgccgacgt gttcgccaag gtgcagagcc tgttcgacgg ctttgacgat caccagaagg
     2821 atggcatcta cgtggagcac aagaacctga atgagctgtc caagcaggcc ttcggcgact
     2881 ttgcactgct gggacgcgtg ctggacggat actatgtgga tgtggtgaat ccagagttca
     2941 acgagcggtt tgccaaggcc aagaccgaca atgccaaggc caagctgaca aaggagaagg
     3001 ataagttcat caagggcgtg cactccctgg cctctctgga gcaggccatc gagcactata
     3061 ccgcaaggca cgacgatgag agcgtgcagg caggcaagct gggacagtac ttcaagcacg
     3121 gcctggccgg agtggacaac cccatccaga agatccacaa caatcacagc accatcaagg
     3181 gctttctgga gagggagcgc cctgcaggag agagagccct gccaaagatc aagtccggca
     3241 agaatcctga gatgacacag ctgaggcagc tgaaggagct gctggataac gccctgaatg
     3301 tggcccactt cgccaagctg ctgaccacaa agaccacact ggacaatcag gatggcaact
     3361 tctatggcga gtttggcgtg ctgtacgacg agctggccaa gatccccacc ctgtataaca
     3421 aggtgagaga ttacctgagc cagaagcctt tctccaccga gaagtacaag ctgaactttg
     3481 gcaatccaac actgctgaat ggctgggacc tgaacaagga gaaggataat ttcggcgtga
     3541 tcctgcagaa ggacggctgc tactatctgg ccctgctgga caaggcccac aagaaggtgt
     3601 ttgataacgc ccctaataca ggcaagagca tctatcagaa gatgatctat aagtacctgg
     3661 aggtgaggaa gcagttcccc aaggtgttct tttccaagga ggccatcgcc atcaactacc
     3721 acccttctaa ggagctggtg gagatcaagg acaagggccg gcagagatcc gacgatgagc
     3781 gcctgaagct gtatcggttt atcctggagt gtctgaagat ccaccctaag tacgataaga
     3841 agttcgaggg cgccatcggc gacatccagc tgtttaagaa ggataagaag ggcagagagg
     3901 tgccaatcag cgagaaggac ctgttcgata agatcaacgg catcttttct agcaagccta
     3961 agctggagat ggaggacttc tttatcggcg agttcaagag gtataaccca agccaggacc
     4021 tggtggatca gtataatatc tacaagaaga tcgactccaa cgataatcgc aagaaggaga
     4081 atttctacaa caatcacccc aagtttaaga aggatctggt gcggtactat tacgagtcta
     4141 tgtgcaagca cgaggagtgg gaggagagct tcgagttttc caagaagctg caggacatcg
     4201 gctgttacgt ggatgtgaac gagctgttta ccgagatcga gacacggaga ctgaattata
     4261 agatctcctt ctgcaacatc aatgccgact acatcgatga gctggtggag cagggccagc
     4321 tgtatctgtt ccagatctac aacaaggact tttccccaaa ggcccacggc aagcccaatc
     4381 tgcacaccct gtacttcaag gccctgtttt ctgaggacaa cctggccgat cctatctata
     4441 agctgaatgg cgaggcccag atcttctaca gaaaggcctc cctggacatg aacgagacaa
     4501 caatccacag ggccggcgag gtgctggaga acaagaatcc cgataatcct aagaagagac
     4561 agttcgtgta cgacatcatc aaggataaga ggtacacaca ggacaagttc atgctgcacg
     4621 tgccaatcac catgaacttt ggcgtgcagg gcatgacaat caaggagttc aataagaagg
     4681 tgaaccagtc tatccagcag tatgacgagg tgaacgtgat cggcatcgat cggggcgaga
     4741 gacacctgct gtacctgacc gtgatcaata gcaagggcga gatcctggag cagtgttccc
     4801 tgaacgacat caccacagcc tctgccaatg gcacacagat gaccacacct taccacaaga
     4861 tcctggataa gagggagatc gagcgcctga acgcccgggt gggatggggc gagatcgaga
     4921 caatcaagga gctgaagtct ggctatctga gccacgtggt gcaccagatc agccagctga
     4981 tgctgaagta caacgccatc gtggtgctgg aggacctgaa tttcggcttt aagaggggcc
     5041 gctttaaggt ggagaagcag atctatcaga acttcgagaa tgccctgatc aagaagctga
     5101 accacctggt gctgaaggac aaggccgacg atgagatcgg ctcttacaag aatgccctgc
     5161 agctgaccaa caatttcaca gatctgaaga gcatcggcaa gcagaccggc ttcctgtttt
     5221 atgtgcccgc ctggaacacc tctaagatcg accctgagac aggctttgtg gatctgctga
     5281 agccaagata cgagaacatc gcccagagcc aggccttctt tggcaagttc gacaagatct
     5341 gctataatgc cgacaaggat tacttcgagt ttcacatcga ctacgccaag tttaccgata
     5401 aggccaagaa tagccgccag atctggacaa tctgttccca cggcgacaag cggtacgtgt
     5461 acgataagac agccaaccag aataagggcg ccgccaaggg catcaacgtg aatgatgagc
     5521 tgaagtccct gttcgcccgc caccacatca acgagaagca gcccaacctg gtcatggaca
     5581 tctgccagaa caatgataag gagtttcaca agtctctgat gtacctgctg aaaaccctgc
     5641 tggccctgcg gtacagcaac gcctcctctg acgaggattt catcctgtcc cccgtggcaa
     5701 acgacgaggg cgtgttcttt aatagcgccc tggccgacga tacacagcct cagaatgccg
     5761 atgccaacgg cgcctaccac atcgccctga agggcctgtg gctgctgaat gagctgaaga
     5821 actccgacga tctgaacaag gtgaagctgg ccatcgacaa tcagacctgg ctgaatttcg
     5881 cccagaacag gaaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     5941 gatcctaccc atacgatgtt ccagattacg cttatcccta cgacgtgcct gattatgcat
     6001 acccatatga tgtccccgac tatgcctaaa gcggccgcac tcctcaggtg caggctgcct
     6061 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     6121 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     6181 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     6241 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     6301 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     6361 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     6421 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     6481 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     6541 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
     6601 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6661 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6721 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt
     6781 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     6841 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     6901 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     6961 gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     7021 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     7081 caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga
     7141 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     7201 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     7261 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     7321 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     7381 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     7441 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     7501 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     7561 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     7621 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     7681 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7741 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     7801 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     7861 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     7921 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     7981 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     8041 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     8101 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     8161 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     8221 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     8281 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     8341 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     8401 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     8461 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     8521 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     8581 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     8641 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     8701 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     8761 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     8821 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     8881 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     8941 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     9001 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     9061 gttccgcgca catttccccg aaaagtgcca cctgggtc
//
