LOCUS       sequence-267376-        8187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter expression plasmid for human codon optimized Nme2Cas9 
            nuclease with C-terminal NLS (SV40) and 3x FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8187)
  AUTHORS   Mahendra C, Christie KA, Osuna BA, Pinilla-Redondo R, Kleinstiver 
            BP, Bondy-Denomy J
  TITLE     Broad-spectrum anti-CRISPR proteins facilitate horizontal gene 
            transfer.
  JOURNAL   Nat Microbiol. 2020 Apr;5(4):620-629. doi: 
            10.1038/s41564-020-0692-2. Epub 2020 Mar 26.
  PUBMED    32218510
REFERENCE   2  (bases 1 to 8187)
  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"; doi: 
            "10.1038/s41564-020-0692-2"; journalName: "Nat Microbiol"; date: 
            "2020-04"; volume: "5"; issue: "4"; pages: "620-629"
COMMENT     Sequence Label: pCAG-hNme2Cas9-NLS(SV40)-3xFLAG  (KAC582)
FEATURES             Location/Qualifiers
     source          1..8187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..275
                     /label=chicken beta-actin promoter
     intron          276..1292
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1216..1238
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1300..1319
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1385..1394
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4643..4663
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4670..4735
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4825..4844)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4890..4945
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4944..4963)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5306..5322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5306..5322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5319..5341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5330..5346
                     /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(5354..5360,5361..5378,5379..5384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5399..5420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5479..5674
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5525..5660
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5680..5814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5717..5736)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5771..5790
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5882..5899)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6053..6641)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6133..6152)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6812..7603,7604..7672))
                     /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     7435..7454
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7673..7777)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        7808..8187
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
       61 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggc
      121 gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
      181 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
      241 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
      301 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
      361 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
      421 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
      481 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg
      541 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg
      601 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
      661 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
      721 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
      781 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
      841 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
      901 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
      961 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
     1021 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc
     1081 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
     1141 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg
     1201 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
     1261 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
     1321 tgtctcatca ttttggcaaa gaattctgca gtcgacggta ccgcgggccc gggatccacc
     1381 ggtcgccacc atggccgcct tcaagcctaa cccaatcaat tacatcctgg gactggacat
     1441 cggaatcgca tccgtgggat gggctatggt ggagatcgac gaggaggaga atcctatccg
     1501 gctgatcgat ctgggcgtga gagtgtttga gagggccgag gtgccaaaga ccggcgattc
     1561 tctggctatg gcccggagac tggcacggag cgtgaggcgc ctgacacgga gaagggcaca
     1621 caggctgctg agggcacgcc ggctgctgaa gagagagggc gtgctgcagg cagcagactt
     1681 cgatgagaat ggcctgatca agagcctgcc aaacaccccc tggcagctga gagcagccgc
     1741 cctggacagg aagctgacac cactggagtg gtctgccgtg ctgctgcacc tgatcaagca
     1801 ccgcggctac ctgagccagc ggaagaacga gggagagaca gcagacaagg agctgggcgc
     1861 cctgctgaag ggagtggcca acaatgccca cgccctgcag accggcgatt tcaggacacc
     1921 tgccgagctg gccctgaata agtttgagaa ggagtccggc cacatcagaa accagagggg
     1981 cgactatagc cacaccttct cccgcaagga tctgcaggcc gagctgatcc tgctgttcga
     2041 gaagcagaag gagtttggca atccacacgt gagcggaggc ctgaaggagg gaatcgagac
     2101 cctgctgatg acacagaggc ctgccctgtc cggcgacgca gtgcagaaga tgctgggaca
     2161 ctgcaccttc gagcctgcag agccaaaggc cgccaagaac acctacacag ccgagcggtt
     2221 tatctggctg acaaagctga acaatctgag aatcctggag cagggatccg agaggccact
     2281 gaccgacaca gagagggcca ccctgatgga tgagccttac cggaagtcta agctgacata
     2341 tgcccaggcc agaaagctgc tgggcctgga ggacaccgcc ttctttaagg gcctgagata
     2401 cggcaaggat aatgccgagg cctccacact gatggagatg aaggcctatc acgccatctc
     2461 tcgcgccctg gagaaggagg gcctgaagga caagaagtcc cccctgaacc tgagctccga
     2521 gctgcaggat gagatcggca ccgccttctc tctgtttaag accgacgagg atatcacagg
     2581 ccgcctgaag gacagggtgc agcctgagat cctggaggcc ctgctgaagc acatctcttt
     2641 cgataagttt gtgcagatca gcctgaaggc cctgagaagg atcgtgccac tgatggagca
     2701 gggcaagcgg tacgacgagg cctgcgccga gatctacggc gatcactatg gcaagaagaa
     2761 cacagaggag aagatctatc tgccccctat ccctgccgac gagatcagaa atcctgtggt
     2821 gctgagggcc ctgtcccagg caagaaaagt gatcaacgga gtggtgcgcc ggtacggatc
     2881 tccagcccgg atccacatcg agaccgccag agaagtgggc aagagcttca aggaccggaa
     2941 ggagatcgag aagagacagg aggagaatcg caaggatcgg gagaaggccg ccgccaagtt
     3001 tagggagtac ttccctaact ttgtgggcga gccaaagtct aaggacatcc tgaagctgcg
     3061 cctgtacgag cagcagcacg gcaagtgtct gtatagcggc aaggagatca atctggtgcg
     3121 gctgaacgag aagggctatg tggagatcga tcacgccctg cctttctcca gaacctggga
     3181 cgattctttt aacaataagg tgctggtgct gggcagcgag aaccagaata agggcaatca
     3241 gacaccatac gagtatttca atggcaagga caactccagg gagtggcagg agttcaaggc
     3301 ccgcgtggag acctctagat ttcccaggag caagaagcag cggatcctgc tgcagaagtt
     3361 cgacgaggat ggctttaagg agtgcaacct gaatgacacc agatacgtga accggttcct
     3421 gtgccagttt gtggccgatc acatcctgct gaccggcaag ggcaagagaa gggtgttcgc
     3481 ctctaatggc cagatcacaa acctgctgag gggattttgg ggactgagga aggtgcgggc
     3541 agagaatgac agacaccacg cactggatgc agtggtggtg gcatgcagca ccgtggcaat
     3601 gcagcagaag atcacaagat tcgtgaggta taaggagatg aacgcctttg acggcaagac
     3661 catcgataag gagacaggca aggtgctgca ccagaagacc cacttccccc agccttggga
     3721 gttctttgcc caggaagtga tgatccgggt gttcggcaag ccagacggca agcctgagtt
     3781 tgaggaggcc gataccccag agaagctgag gacactgctg gcagagaagc tgtctagcag
     3841 gccagaggca gtgcacgagt acgtgacccc actgttcgtg tccagggcac ccaatcggaa
     3901 gatgtctggc gcccacaagg acacactgag aagcgccaag aggtttgtga agcacaacga
     3961 gaagatctcc gtgaagagag tgtggctgac cgagatcaag ctggccgatc tggagaacat
     4021 ggtgaattac aagaacggca gggagatcga gctgtatgag gccctgaagg caaggctgga
     4081 ggcctacgga ggaaatgcca agcaggcctt cgacccaaag gataacccct tttataagaa
     4141 gggaggacag ctggtgaagg ccgtgcgggt ggagaagacc caggagagcg gcgtgctgct
     4201 gaataagaag aacgcctaca caatcgccga caatggcgat atggtgagag tggacgtgtt
     4261 ctgtaaggtg gataagaagg gcaagaatca gtactttatc gtgcctatct atgcctggca
     4321 ggtggccgag aacatcctgc cagacatcga ttgcaagggc tacagaatcg acgatagcta
     4381 tacattctgt ttttccctgc acaagtatga cctgatcgcc ttccagaagg atgagaagtc
     4441 caaggtggag tttgcctact atatcaattg cgactcctct aacggcaggt tctacctggc
     4501 ctggcacgat aagggcagca aggagcagca gtttcgcatc tccacccaga atctggtgct
     4561 gatccagaag tatcaggtga acgagctggg caaggagatc aggccatgtc ggctgaagaa
     4621 gcgcccaccc gtgcggggat cccccaagaa gaagaggaaa gtctcgagcg actacaaaga
     4681 ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg acaagtaaag
     4741 cggccgcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
     4801 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
     4861 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     4921 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     4981 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     5041 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     5101 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     5161 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     5221 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     5281 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5341 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5401 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5461 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     5521 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5581 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     5641 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     5701 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5761 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     5821 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5881 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5941 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6001 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6061 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6121 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6181 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6241 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6301 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6361 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6421 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6481 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6541 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6601 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6661 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6721 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6781 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6841 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6901 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6961 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7021 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7081 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7141 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7201 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7261 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7321 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7381 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7441 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7501 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7561 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7621 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7681 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7741 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7801 ctgggtcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     7861 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     7921 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     7981 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     8041 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     8101 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     8161 tacgtattag tcatcgctat taccatg
//
