LOCUS       sequence-364684-        8105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human expression plasmid for SaCas9 KKH variant: 
            CAG-humanSaCas9(E782K/N968K/R1015H)-NLS-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8105)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, 
            Joung JK
  TITLE     Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 
            by modifying PAM recognition.
  JOURNAL   Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404.
  PUBMED    26524662
REFERENCE   2  (bases 1 to 8105)
  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. 2015 Nov 2. doi: 10.1038/nbt.3404."
COMMENT     Sequence Label: MSP1830
FEATURES             Location/Qualifiers
     source          1..8105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1771..1780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4945..4965
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4972..5037
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5127..5146)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5192..5247
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5246..5265)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5608..5624)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5608..5624)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5621..5643)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5632..5648
                     /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(5656..5662,5663..5680,5681..5686))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5701..5722
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5781..5976
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5827..5962
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5982..6116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6019..6038)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6073..6092
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6184..6201)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6355..6943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6435..6454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7114..7905,7906..7974))
                     /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     7737..7756
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7975..8079)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggsgc gccvggsggg gsggggsggg gsgrggggsg
      541 gggsggggsg rggsggrgrg gkgsggsggc rgccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gkgggggtgc cgggcggggc
     1261 ggggccgccy cgggccgggg rgggctcggg ggrggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tctgcagtcg
     1741 acggtaccgc gggcccggga tccaccggtc gccaccatgg ggaaacggaa ctacatcctg
     1801 gggcttgaca ttgggataac cagcgttggc tacggaatta ttgattatga gacacgcgat
     1861 gtgattgacg ccggggttag gctgttcaaa gaggccaacg ttgaaaacaa cgagggaaga
     1921 cggagtaagc gcggagcaag aagactcaag cgcagacgga gacatcggat tcagagggtg
     1981 aaaaagctgc tcttcgatta caatctcctg accgatcata gtgagctgag cggaatcaac
     2041 ccctacgagg cgcgagtgaa agggctttcc cagaagctgt ccgaagagga gttctccgcc
     2101 gcgttgctgc acctggccaa acggaggggg gttcacaatg taaacgaagt ggaggaggac
     2161 acgggcaatg aacttagtac gaaagaacag atcagtagga actctaaggc tctcgaagag
     2221 aaatacgtcg ctgagttgca gcttgagaga ctgaaaaaag acggcgaagt acgcggatct
     2281 attaataggt tcaagacttc agattacgta aaggaagcca agcagctcct gaaagtacag
     2341 aaagcgtacc atcagctcga tcagagcttc atcgatacct acatagattt gctggagaca
     2401 cggaggacat actacgaggg cccaggggaa ggatctcctt ttgggtggaa ggacatcaag
     2461 gaatggtacg agatgcttat gggacattgt acatattttc cggaggagct caggagcgtc
     2521 aagtacgcct acaatgccga cctgtacaat gccctcaatg acctcaataa cctcgtgatt
     2581 accagggacg agaacgagaa gctggagtac tatgaaaagt tccagattat cgagaatgtg
     2641 tttaagcaga agaagaagcc gacacttaag cagattgcaa aggaaatcct cgtgaatgag
     2701 gaagatatca agggatacag agtgacaagt acaggcaagc ccgagttcac aaatctgaag
     2761 gtgtaccacg atattaagga cataaccgca cgaaaggaga taatcgaaaa cgctgagctc
     2821 ctcgatcaga tcgcaaaaat tcttaccatc taccagtcta gtgaggacat tcaggaggaa
     2881 ctgactaatc tgaacagtga gctcacccaa gaggaaattg agcagatttc aaacctgaaa
     2941 ggctacaccg ggacgcacaa tctgagcctc aaagcaatca acctcattct ggatgaactt
     3001 tggcacacaa atgacaacca aattgccata ttcaaccgcc tgaaactggt gccaaaaaaa
     3061 gtggatctgt cacagcaaaa ggaaatccct acaaccttgg ttgacgattt tattctgtcc
     3121 cccgttgtca agcggagctt catccagtca atcaaggtga tcaatgccat cattaaaaaa
     3181 tacggattgc caaacgatat aattatcgag cttgcacgag agaagaactc aaaggacgcc
     3241 cagaagatga ttaacgaaat gcagaagcgc aaccgccaga caaacgaacg catagaggaa
     3301 attataagaa caaccggcaa agagaatgcc aagtatctga tcgagaaaat caagctgcac
     3361 gacatgcaag aaggcaagtg cctgtactct ctggaagcta tcccactcga agatctgctg
     3421 aataatccat tcaattacga ggtggaccac atcatcccta gatccgtaag ctttgacaat
     3481 tccttcaata acaaagttct ggttaaacag gaggaaaatt ctaaaaaagg gaaccggacc
     3541 ccgttccagt acctgagctc cagtgacagc aagattagct acgagacttt taagaaacat
     3601 attctgaatc tggccaaagg caaaggcagg atcagcaaga ccaagaagga gtacctcctc
     3661 gaagaacgcg acattaacag atttagtgtg cagaaagatt tcatcaaccg aaaccttgtc
     3721 gatactcggt acgccacgag aggcctgatg aatctcctca ggagctactt ccgcgtcaat
     3781 aatctggacg ttaaagtcaa gagcataaat gggggattca ccagctttct gaggagaaag
     3841 tggaagttta agaaggaacg aaacaaagga tacaagcacc atgctgagga tgctttgatc
     3901 atcgctaacg cggactttat ctttaaggaa tggaaaaagc tggataaggc aaagaaagtg
     3961 atggaaaacc agatgttcga ggagaagcag gcagagtcaa tgcctgagat cgagacagag
     4021 caggaataca aggaaatttt catcacccct catcagatta aacacataaa ggacttcaaa
     4081 gactataaat actctcatag ggtggacaaa aaacccaatc gcaagctcat taatgacacc
     4141 ctgtactcaa cacggaagga tgataaaggt aataccttga ttgtgaataa tcttaatgga
     4201 ttgtatgaca aagataacga caagctcaag aagctgatca acaagtctcc agagaagctc
     4261 cttatgtatc accacgaccc acagacttat cagaaattga aactgatcat ggagcaatac
     4321 ggggatgaga agaacccact ctacaaatat tatgaggaaa caggtaatta cctgaccaag
     4381 tactccaaga aggataacgg accagtgatc aaaaagataa agtactatgg caacaaactt
     4441 aatgcgcatt tggacataac tgacgattac cccaattctc gaaacaaggt tgtgaagctc
     4501 tccctgaagc cttatagatt tgacgtgtac ctggataatg gggtttataa attcgtcacc
     4561 gtgaaaaatc tggacgtgat caaaaaggag aactattatg aagtaaactc aaagtgctat
     4621 gaggaggcga agaagctgaa gaagatctcc aatcaggccg agttcatcgc ttccttctat
     4681 aagaacgatc tcatcaagat caatggagag ctttatcgcg tcattggtgt gaacaatgac
     4741 ttgctgaaca ggatcgaagt caatatgata gacattacct accgggagta tctcgaaaac
     4801 atgaatgata aacggccgcc tcacatcatc aagacaatcg catctaaaac tcagtcaata
     4861 aaaaagtact ctaccgatat cctggggaat ctctatgaag tgaagtcaaa gaagcaccca
     4921 caaatcatta aaaaaggtgg atcccccaag aagaagagga aagtctcgag cgactacaaa
     4981 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagtaa
     5041 agcggccgca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat
     5101 gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc
     5161 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     5221 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     5281 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc
     5341 atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     5401 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     5461 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     5521 ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac
     5581 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5641 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5701 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5761 cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     5821 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     5881 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     5941 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     6001 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     6061 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     6121 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6181 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6241 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6301 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6361 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6421 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6481 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6541 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6601 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6661 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6721 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6781 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6841 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6901 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6961 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7021 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7081 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7141 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7201 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7261 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7321 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7381 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7441 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7501 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7561 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7621 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7681 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7741 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7801 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7861 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7921 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7981 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8041 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8101 acctg
//
