LOCUS       sequence-227326-        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"
     CDS             13..33
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             34..123
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             136..162
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     protein_bind    complement(200..224)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    269..403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(293..312)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     347..366
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(516..536)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(516..534)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(555..571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(555..571)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(568..590)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    579..595
                     /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(603..609,610..627,628..633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    648..669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(786..803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(957..1545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1037..1056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1716..2507,2508..2576))
                     /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     2339..2358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2577..2681)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2707..3162)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2844..2865)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3056..3075
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3233..4217
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        4252..4270
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4252..4269
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    4325..4349
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ggcaccggcg ggcccaagaa gaagaggaag gtatacccat acgatgttcc tgactatgcg
       61 ggctatccct atgacgtccc ggactatgca ggatcgtatc cttatgacgt tccagattac
      121 gctggatccg ccgctccggc agctaagaaa aagaaactgg atttcgaatc cggataagcg
      181 gccgcctcga gatatctaga cccagctttc ttgtacaaag tggttgatcc tctcgagcct
      241 ctagaactat agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt
      301 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
      361 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
      421 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc
      481 gccaatgcat tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc
      541 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      601 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      661 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      721 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      781 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      841 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      901 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      961 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1021 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1081 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1141 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1201 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1261 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1321 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1381 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1441 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1501 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1561 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1621 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1681 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1741 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1801 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1861 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1921 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1981 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2041 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2101 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2161 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2221 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2281 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2341 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2401 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2461 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2521 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2581 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2641 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2701 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2761 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2821 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2881 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2941 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3001 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3061 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3121 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     3181 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagtcgaccg
     3241 ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc tggacctgtg
     3301 ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc tggaccccag
     3361 ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggact tggcactgtg
     3421 ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc ttggccctgt
     3481 gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat tggctatatg
     3541 ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc tattggccag
     3601 gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt tttcctattg
     3661 acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct tcctaatacc
     3721 gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt cctattgacg
     3781 tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca attacggtaa
     3841 atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat tgacgtcaat
     3901 gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt caatgacggt
     3961 aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa gtacattact
     4021 attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg cggtaaatgg
     4081 cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca atgacgcaaa
     4141 tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc tatataagca
     4201 atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt gatttaggtg
     4261 acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgattc gaattcaagg
     4321 atcaacaagt ttgtacaaaa aagcaggctg gcgccaccat gatggccgcc ttcaagccta
     4381 acccaatcaa ttacatcctg ggactggaca tcggaatcgc atccgtggga tgggctatgg
     4441 tggagatcga cgaggaggag aatcctatcc ggctgatcga tctgggcgtg agagtgtttg
     4501 agagggccga ggtgccaaag accggcgatt ctctggctat ggcccggaga ctggcacgga
     4561 gcgtgaggcg cctgacacgg agaagggcac acaggctgct gagggcacgc cggctgctga
     4621 agagagaggg cgtgctgcag gcagcagact tcgatgagaa tggcctgatc aagagcctgc
     4681 caaacacccc ctggcagctg agagcagccg ccctggacag gaagctgaca ccactggagt
     4741 ggtctgccgt gctgctgcac ctgatcaagc accgcggcta cctgagccag cggaagaacg
     4801 agggagagac agcagacaag gagctgggcg ccctgctgaa gggagtggcc aacaatgccc
     4861 acgccctgca gaccggcgat ttcaggacac ctgccgagct ggccctgaat aagtttgaga
     4921 aggagtccgg ccacatcaga aaccagaggg gcgactatag ccacaccttc tcccgcaagg
     4981 atctgcaggc cgagctgatc ctgctgttcg agaagcagaa ggagtttggc aatccacacg
     5041 tgagcggagg cctgaaggag ggaatcgaga ccctgctgat gacacagagg cctgccctgt
     5101 ccggcgacgc agtgcagaag atgctgggac actgcacctt cgagcctgca gagccaaagg
     5161 ccgccaagaa cacctacaca gccgagcggt ttatctggct gacaaagctg aacaatctga
     5221 gaatcctgga gcagggatcc gagaggccac tgaccgacac agagagggcc accctgatgg
     5281 atgagcctta ccggaagtct aagctgacat atgcccaggc cagaaagctg ctgggcctgg
     5341 aggacaccgc cttctttaag ggcctgagat acggcaagga taatgccgag gcctccacac
     5401 tgatggagat gaaggcctat cacgccatct ctcgcgccct ggagaaggag ggcctgaagg
     5461 acaagaagtc ccccctgaac ctgagctccg agctgcagga tgagatcggc accgccttct
     5521 ctctgtttaa gaccgacgag gatatcacag gccgcctgaa ggacagggtg cagcctgaga
     5581 tcctggaggc cctgctgaag cacatctctt tcgataagtt tgtgcagatc agcctgaagg
     5641 ccctgagaag gatcgtgcca ctgatggagc agggcaagcg gtacgacgag gcctgcgccg
     5701 agatctacgg cgatcactat ggcaagaaga acacagagga gaagatctat ctgcccccta
     5761 tccctgccga cgagatcaga aatcctgtgg tgctgagggc cctgtcccag gcaagaaaag
     5821 tgatcaacgg agtggtgcgc cggtacggat ctccagcccg gatccacatc gagaccgcca
     5881 gagaagtggg caagagcttc aaggaccgga aggagatcga gaagagacag gaggagaatc
     5941 gcaaggatcg ggagaaggcc gccgccaagt ttagggagta cttccctaac tttgtgggcg
     6001 agccaaagtc taaggacatc ctgaagctgc gcctgtacga gcagcagcac ggcaagtgtc
     6061 tgtatagcgg caaggagatc aatctggtgc ggctgaacga gaagggctat gtggagatcg
     6121 atcacgccct gcctttctcc agaacctggg acgattcttt taacaataag gtgctggtgc
     6181 tgggcagcga gaaccagaat aagggcaatc agacaccata cgagtatttc aatggcaagg
     6241 acaactccag ggagtggcag gagttcaagg cccgcgtgga gacctctaga tttcccagga
     6301 gcaagaagca gcggatcctg ctgcagaagt tcgacgagga tggctttaag gagtgcaacc
     6361 tgaatgacac cagatacgtg aaccggttcc tgtgccagtt tgtggccgat cacatcctgc
     6421 tgaccggcaa gggcaagaga agggtgttcg cctctaatgg ccagatcaca aacctgctga
     6481 ggggattttg gggactgagg aaggtgcggg cagagaatga cagacaccac gcactggatg
     6541 cagtggtggt ggcatgcagc accgtggcaa tgcagcagaa gatcacaaga ttcgtgaggt
     6601 ataaggagat gaacgccttt gacggcaaga ccatcgataa ggagacaggc aaggtgctgc
     6661 accagaagac ccacttcccc cagccttggg agttctttgc ccaggaagtg atgatccggg
     6721 tgttcggcaa gccagacggc aagcctgagt ttgaggaggc cgatacccca gagaagctga
     6781 ggacactgct ggcagagaag ctgtctagca ggccagaggc agtgcacgag tacgtgaccc
     6841 cactgttcgt gtccagggca cccaatcgga agatgtctgg cgcccacaag gacacactga
     6901 gaagcgccaa gaggtttgtg aagcacaacg agaagatctc cgtgaagaga gtgtggctga
     6961 ccgagatcaa gctggccgat ctggagaaca tggtgaatta caagaacggc agggagatcg
     7021 agctgtatga ggccctgaag gcaaggctgg aggcctacgg aggaaatgcc aagcaggcct
     7081 tcgacccaaa ggataacccc ttttataaga agggaggaca gctggtgaag gccgtgcggg
     7141 tggagaagac ccaggagagc ggcgtgctgc tgaataagaa gaacgcctac acaatcgccg
     7201 acaatggcga tatggtgaga gtggacgtgt tctgtaaggt ggataagaag ggcaagaatc
     7261 agtactttat cgtgcctatc tatgcctggc aggtggccga gaacatcctg ccagacatcg
     7321 attgcaaggg ctacagaatc gacgatagct atacattctg tttttccctg cacaagtatg
     7381 acctgatcgc cttccagaag gatgagaagt ccaaggtgga gtttgcctac tatatcaatt
     7441 gcgactcctc taacggcagg ttctacctgg cctggcacga taagggcagc aaggagcagc
     7501 agtttcgcat ctccacccag aatctggtgc tgatccagaa gtatcaggtg aacgagctgg
     7561 gcaaggagat caggccatgt cggctgaaga agcgcccacc cgtgcgg
//
