LOCUS       sequence-233417-        7607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nme2Cas9 CMV-driven mammalian expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7607)
  AUTHORS   Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, 
            Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
  TITLE     A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo 
            Genome Editing.
  JOURNAL   Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003.
  PUBMED    30581144
REFERENCE   2  (bases 1 to 7607)
  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: "Mol Cell. 
            2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003."
COMMENT     Sequence Label: Nme2Cas9_pCDest
FEATURES             Location/Qualifiers
     source          1..7607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        101..1085
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1120..1138
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1120..1137
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1193..1217
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4488..4508
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4509..4598
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4611..4637
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     protein_bind    complement(4675..4699)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4744..4878
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4768..4787)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4822..4841
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4991..5011)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4991..5009)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5030..5046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5030..5046)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5043..5065)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5054..5070
                     /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(5078..5084,5085..5102,5103..5108))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5123..5144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5261..5278)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5432..6020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5512..5531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6191..6982,6983..7051))
                     /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     6814..6833
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7052..7156)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(7182..7607,1..30))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7319..7340)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7531..7550
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
       61 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc aattcaatat
      121 ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc aattcaatat
      181 ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc aattcaatat
      241 ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc aactggggag
      301 gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc caagtccgcc
      361 atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc aggatcaata
      421 tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt tcaatactat
      481 gtattggccc tatgccatat agtattccat atatgggttt tcctattgac gtagatagcc
      541 cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc ccatagccac
      601 tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc atatgggcgg
      661 tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat ggcccgcctg
      721 gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg gatggctcat
      781 tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa atggcccact
      841 tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat tggaagtacg
      901 ccagggtaca ttggcagtac tcccattgac gtcaatggcg gtaaatggcc cgcgatggct
      961 gccaagtaca tccccattga cgtcaatggg gaggggcaat gacgcaaatg ggcgttccat
     1021 tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta tataagcaat gctcgtttag
     1081 ggaaccgcca ttctgcctgg ggacgtcgga gcaagcttga tttaggtgac actatagaat
     1141 acaagctact tgttcttttt gcaggatccc atcgattcga attcaaggat caacaagttt
     1201 gtacaaaaaa gcaggctggc gccaccatga tggccgcctt caagcctaac ccaatcaatt
     1261 acatcctggg actggacatc ggaatcgcat ccgtgggatg ggctatggtg gagatcgacg
     1321 aggaggagaa tcctatccgg ctgatcgatc tgggcgtgag agtgtttgag agggccgagg
     1381 tgccaaagac cggcgattct ctggctatgg cccggagact ggcacggagc gtgaggcgcc
     1441 tgacacggag aagggcacac aggctgctga gggcacgccg gctgctgaag agagagggcg
     1501 tgctgcaggc agcagacttc gatgagaatg gcctgatcaa gagcctgcca aacaccccct
     1561 ggcagctgag agcagccgcc ctggacagga agctgacacc actggagtgg tctgccgtgc
     1621 tgctgcacct gatcaagcac cgcggctacc tgagccagcg gaagaacgag ggagagacag
     1681 cagacaagga gctgggcgcc ctgctgaagg gagtggccaa caatgcccac gccctgcaga
     1741 ccggcgattt caggacacct gccgagctgg ccctgaataa gtttgagaag gagtccggcc
     1801 acatcagaaa ccagaggggc gactatagcc acaccttctc ccgcaaggat ctgcaggccg
     1861 agctgatcct gctgttcgag aagcagaagg agtttggcaa tccacacgtg agcggaggcc
     1921 tgaaggaggg aatcgagacc ctgctgatga cacagaggcc tgccctgtcc ggcgacgcag
     1981 tgcagaagat gctgggacac tgcaccttcg agcctgcaga gccaaaggcc gccaagaaca
     2041 cctacacagc cgagcggttt atctggctga caaagctgaa caatctgaga atcctggagc
     2101 agggatccga gaggccactg accgacacag agagggccac cctgatggat gagccttacc
     2161 ggaagtctaa gctgacatat gcccaggcca gaaagctgct gggcctggag gacaccgcct
     2221 tctttaaggg cctgagatac ggcaaggata atgccgaggc ctccacactg atggagatga
     2281 aggcctatca cgccatctct cgcgccctgg agaaggaggg cctgaaggac aagaagtccc
     2341 ccctgaacct gagctccgag ctgcaggatg agatcggcac cgccttctct ctgtttaaga
     2401 ccgacgagga tatcacaggc cgcctgaagg acagggtgca gcctgagatc ctggaggccc
     2461 tgctgaagca catctctttc gataagtttg tgcagatcag cctgaaggcc ctgagaagga
     2521 tcgtgccact gatggagcag ggcaagcggt acgacgaggc ctgcgccgag atctacggcg
     2581 atcactatgg caagaagaac acagaggaga agatctatct gccccctatc cctgccgacg
     2641 agatcagaaa tcctgtggtg ctgagggccc tgtcccaggc aagaaaagtg atcaacggag
     2701 tggtgcgccg gtacggatct ccagcccgga tccacatcga gaccgccaga gaagtgggca
     2761 agagcttcaa ggaccggaag gagatcgaga agagacagga ggagaatcgc aaggatcggg
     2821 agaaggccgc cgccaagttt agggagtact tccctaactt tgtgggcgag ccaaagtcta
     2881 aggacatcct gaagctgcgc ctgtacgagc agcagcacgg caagtgtctg tatagcggca
     2941 aggagatcaa tctggtgcgg ctgaacgaga agggctatgt ggagatcgat cacgccctgc
     3001 ctttctccag aacctgggac gattctttta acaataaggt gctggtgctg ggcagcgaga
     3061 accagaataa gggcaatcag acaccatacg agtatttcaa tggcaaggac aactccaggg
     3121 agtggcagga gttcaaggcc cgcgtggaga cctctagatt tcccaggagc aagaagcagc
     3181 ggatcctgct gcagaagttc gacgaggatg gctttaagga gtgcaacctg aatgacacca
     3241 gatacgtgaa ccggttcctg tgccagtttg tggccgatca catcctgctg accggcaagg
     3301 gcaagagaag ggtgttcgcc tctaatggcc agatcacaaa cctgctgagg ggattttggg
     3361 gactgaggaa ggtgcgggca gagaatgaca gacaccacgc actggatgca gtggtggtgg
     3421 catgcagcac cgtggcaatg cagcagaaga tcacaagatt cgtgaggtat aaggagatga
     3481 acgcctttga cggcaagacc atcgataagg agacaggcaa ggtgctgcac cagaagaccc
     3541 acttccccca gccttgggag ttctttgccc aggaagtgat gatccgggtg ttcggcaagc
     3601 cagacggcaa gcctgagttt gaggaggccg ataccccaga gaagctgagg acactgctgg
     3661 cagagaagct gtctagcagg ccagaggcag tgcacgagta cgtgacccca ctgttcgtgt
     3721 ccagggcacc caatcggaag atgtctggcg cccacaagga cacactgaga agcgccaaga
     3781 ggtttgtgaa gcacaacgag aagatctccg tgaagagagt gtggctgacc gagatcaagc
     3841 tggccgatct ggagaacatg gtgaattaca agaacggcag ggagatcgag ctgtatgagg
     3901 ccctgaaggc aaggctggag gcctacggag gaaatgccaa gcaggccttc gacccaaagg
     3961 ataacccctt ttataagaag ggaggacagc tggtgaaggc cgtgcgggtg gagaagaccc
     4021 aggagagcgg cgtgctgctg aataagaaga acgcctacac aatcgccgac aatggcgata
     4081 tggtgagagt ggacgtgttc tgtaaggtgg ataagaaggg caagaatcag tactttatcg
     4141 tgcctatcta tgcctggcag gtggccgaga acatcctgcc agacatcgat tgcaagggct
     4201 acagaatcga cgatagctat acattctgtt tttccctgca caagtatgac ctgatcgcct
     4261 tccagaagga tgagaagtcc aaggtggagt ttgcctacta tatcaattgc gactcctcta
     4321 acggcaggtt ctacctggcc tggcacgata agggcagcaa ggagcagcag tttcgcatct
     4381 ccacccagaa tctggtgctg atccagaagt atcaggtgaa cgagctgggc aaggagatca
     4441 ggccatgtcg gctgaagaag cgcccacccg tgcggggcac cggcgggccc aagaagaaga
     4501 ggaaggtata cccatacgat gttcctgact atgcgggcta tccctatgac gtcccggact
     4561 atgcaggatc gtatccttat gacgttccag attacgctgg atccgccgct ccggcagcta
     4621 agaaaaagaa actggatttc gaatccggat aagcggccgc ctcgagatat ctagacccag
     4681 ctttcttgta caaagtggtt gatcctctcg agcctctaga actatagtga gtcgtattac
     4741 gtagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     4801 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     4861 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     4921 gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtaccca
     4981 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     5041 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc gggagcataa
     5101 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5161 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5221 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5281 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5341 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5401 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5461 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5521 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5581 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5641 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5701 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5761 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5821 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5881 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5941 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6001 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6061 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6121 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6181 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6241 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6301 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6361 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6421 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6481 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6541 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6601 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6661 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6721 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6781 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6841 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6901 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6961 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7021 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7081 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7141 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     7201 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     7261 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     7321 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     7381 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     7441 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     7501 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     7561 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccg
//
