LOCUS       sequence-112658-        8234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human expression vector for S. thermophilus 1 Cas9: 
            CAG-humanSt1Cas9-NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8234)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 8234)
  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: "Nature. 
            2015 Jun 22. doi: 10.1038/nature14592."
COMMENT     Sequence Label: MSP1594
FEATURES             Location/Qualifiers
     source          1..8234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1763..1772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1769..5134
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus 
                     thermophilus Type II CRISPR/Cas system (Esvelt et al., 
                     2013)"
                     /label=St1Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MGSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNL
                     VRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNE
                     ELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTY
                     GQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTG
                     KRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL
                     LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRI
                     DKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD
                     GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQK
                     TTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNE
                     DDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGER
                     CLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQAL
                     DSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRV
                     VLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLN
                     LWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFS
                     YQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKS
                     KFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNG
                     PEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADL
                     QFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSR
                     TMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGN
                     QHIIKNEGDKPKLDF"
     CDS             5147..5167
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5256..5275)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5321..5376
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5375..5394)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5737..5753)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5737..5753)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5750..5772)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5761..5777
                     /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(5785..5791,5792..5809,5810..5815))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5830..5851
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5910..6105
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5956..6091
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6111..6245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6148..6167)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6202..6221
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6313..6330)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6484..7072)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6564..6583)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7243..8034,8035..8103))
                     /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     7866..7885
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8104..8208)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc
     1741 gcgggcccgg gatccaccgg tcgccaccat gggcagcgac ctggtgctgg gcctggacat
     1801 cggcatcggc agcgtgggcg tgggcatcct gaacaaggtg accggcgaga tcatccacaa
     1861 gaacagtcgc atcttccctg ctgctcaggc tgagaacaac ctggtgcgcc gcaccaaccg
     1921 ccagggtcgc cggcttgctc gccgcaagaa gcaccggcgc gtgcgcctga accgcctgtt
     1981 cgaggagagc ggcctgatca ccgacttcac caagatcagc atcaacctga acccctacca
     2041 gctgcgcgtg aagggcctga ccgacgagct gagcaacgag gagctgttca tcgccctgaa
     2101 gaacatggtg aagcaccgcg gcatcagcta cctggacgac gccagcgacg acggcaacag
     2161 cagcgtgggc gactacgccc agatcgtgaa ggagaacagc aagcagctgg agaccaagac
     2221 ccccggccag atccagctgg agcgctacca gacctacggc cagctgcgcg gcgacttcac
     2281 cgtggagaag gacggcaaga agcaccgcct gatcaacgtg ttccccacca gcgcctaccg
     2341 cagcgaggcc ctgcgcatcc tgcagaccca gcaggagttc aacccccaga tcaccgacga
     2401 gttcatcaac cgctacctgg agatcctgac cggcaagcgc aagtactacc acggccccgg
     2461 caacgagaag agccgcaccg actacggccg ctaccgcacc agcggcgaga ccctggacaa
     2521 catcttcggc atcctgatcg gcaagtgcac cttctacccc gacgagttcc gcgccgccaa
     2581 ggccagctac accgcccagg agttcaatct gctgaacgac ctgaacaacc tgaccgtgcc
     2641 caccgagacc aagaagctga gcaaggagca gaagaaccag atcatcaact acgtgaagaa
     2701 cgagaaggct atgggccccg ccaagctgtt caagtacatc gccaagctgc tgagctgcga
     2761 cgtggccgac atcaagggct accgcatcga caagagcggc aaggccgaga tccacacctt
     2821 cgaggcctac cgcaagatga agaccctgga gaccctggac atcgagcaga tggaccgaga
     2881 gaccctggac aagctggcct acgtgctgac cctgaacacc gagcgcgagg gcatccagga
     2941 ggccctggag cacgagttcg ccgacggcag cttcagccag aaacaggtgg acgagctggt
     3001 gcagttccgc aaggccaaca gcagcatctt cggcaagggc tggcacaact tcagcgtgaa
     3061 gctgatgatg gagctgatcc ccgagctgta cgagaccagc gaggagcaga tgaccatcct
     3121 gacccgcctg ggcaagcaga agaccaccag cagcagcaac aagaccaagt acatcgacga
     3181 gaagctgctg accgaggaga tctacaaccc cgtggtggcc aagagcgtgc gccaggccat
     3241 caagatcgtg aacgccgcca tcaaggagta cggcgacttc gacaacatcg tgatcgagat
     3301 ggcccgcgag accaacgagg acgacgagaa gaaggccatc cagaagatcc agaaggccaa
     3361 caaggacgag aaggacgccg ccatgctgaa ggccgccaac cagtacaacg gcaaggccga
     3421 gctgccccac agcgtgttcc acggccacaa gcagctggcc accaagatcc gcctgtggca
     3481 ccagcagggc gagcgctgcc tgtacaccgg caagaccatc agcatccacg acctgatcaa
     3541 caacagcaac cagttcgagg tggaccacat cctgcccctg agcatcacct tcgacgacag
     3601 cctggccaac aaggtgctgg tgtacgccac cgccaaccag gagaagggcc agcgcacccc
     3661 ctaccaggcc ctggacagca tggacgacgc ctggagcttc cgcgagctga aggccttcgt
     3721 gcgcgagagc aagaccctga gcaacaagaa gaaggagtat ctgctgaccg aggaggacat
     3781 cagcaagttc gacgtgcgca agaagttcat cgagcgcaac ctggtggaca cccgctacgc
     3841 cagccgcgtg gtgctgaacg ccctgcagga gcacttccgc gcccacaaga tcgacaccaa
     3901 ggtgagcgtg gtgcgcggcc agttcaccag ccagctgcgc cgccactggg gcatcgagaa
     3961 gacccgcgac acctaccacc accacgccgt ggacgccctg atcattgcgg cttctagcca
     4021 gctgaacctg tggaagaagc agaagaacac cctggtgagc tacagcgagg accagctgct
     4081 ggacatcgag accggcgagc tgatcagcga cgacgagtac aaggagagcg tgttcaaggc
     4141 cccctaccag cacttcgtgg acaccctgaa gagcaaggag ttcgaggaca gcatcctgtt
     4201 cagctaccag gtggacagca agttcaaccg caagatcagc gacgccacca tctacgccac
     4261 ccgccaggcc aaggtgggca aggacaaggc cgacgagacc tacgtgctgg gcaagatcaa
     4321 ggacatctac acccaggacg gctacgacgc cttcatgaag atctacaaga aggacaagag
     4381 caagttcctg atgtaccgcc acgaccccca gaccttcgag aaggtgatcg agcccatcct
     4441 ggagaactac cccaacaagc agatcaacga gaaaggcaag gaggtgccct gcaacccctt
     4501 cctgaagtac aaggaggagc acggctacat ccgcaagtac agcaagaagg gcaacggccc
     4561 cgagatcaag agcctgaagt actacgacag caagctgggc aaccacatcg acatcacccc
     4621 caaggacagc aacaacaagg tggtgctgca gagcgtgagc ccctggcgcg ccgacgtgta
     4681 cttcaacaag accaccggca agtacgagat cctggggctg aagtacgccg atctgcagtt
     4741 tgagaaaggc acaggcacct acaagatcag ccaggagaag tacaacgaca tcaagaagaa
     4801 ggagggcgtg gacagcgaca gcgagttcaa gttcaccctg tacaagaacg accttctgct
     4861 ggtgaaggac accgagacca aggagcaaca gctgttccgc ttcctgagcc gcaccatgcc
     4921 caagcagaag cactacgtgg agctgaagcc ctacgacaag cagaagttcg agggcggcga
     4981 ggccctgatc aaggtgctgg gcaacgtggc caacagcggc cagtgcaaga agggcctggg
     5041 caagagcaac atcagcatct acaaggtgcg caccgacgtg ctgggcaacc agcacatcat
     5101 caagaacgag ggcgacaagc ccaagttgga cttcagcagg gctgacccca agaagaagag
     5161 gaaggtgtga gcggccgcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt
     5221 gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat
     5281 ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc
     5341 attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca
     5401 aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg
     5461 ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct
     5521 gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt
     5581 tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc
     5641 agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct cttatgaaga
     5701 tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5761 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5821 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5881 gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg
     5941 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6001 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6061 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta
     6121 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     6181 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     6241 ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6301 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6361 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6421 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6481 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6541 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6601 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6661 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6721 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6781 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6841 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6901 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     6961 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7021 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7081 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7141 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7201 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7261 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7321 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7381 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7441 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7501 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7561 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7621 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7681 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7741 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7801 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7861 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7921 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7981 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8041 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8101 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8161 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8221 aaaagtgcca cctg
//
