LOCUS       sequence-449970-        8841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Nuclease active S. uberis Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8841)
  AUTHORS   Butterfield GL, Rohm D, Roberts A, Nethery MA, Rizzo AJ, Morone DJ, 
            Garnier L, Iglesias N, Barrangou R, Gersbach CA
  TITLE     Characterization of diverse Cas9 orthologs for genome and epigenome 
            editing.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Mar 18;122(11):e2417674122. doi: 
            10.1073/pnas.2417674122. Epub 2025 Mar 12.
  PUBMED    40073054
REFERENCE   2  (bases 1 to 8841)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-03-18"; volume: "122(11):e2417674122. 
            doi"; pages: " 10.1073/pnas.2417674122. Epub 2025 Mar 12"
COMMENT     Sequence Label: pcDNA-Sub-Cas9
FEATURES             Location/Qualifiers
     source          1..8841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             982..1002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4390..4410
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4432..4449)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4438..4662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4708..5136
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4795..4814)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5005..5026
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5145..5165)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5150..5479
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5330..5465
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5392..5411
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5546..6340
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5600..5619)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6210..6229
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6514..6635
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6551..6570)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6605..6624
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6684..6700)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6684..6700)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6697..6719)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6708..6724
                     /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(6732..6738,6739..6756,6757..6762))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6777..6798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6915..6932)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7086..7674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7166..7185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7845..8636,8637..8705))
                     /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     8468..8487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8706..8810)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agcacgttaa ccccaagaag aagaggaagg tgggccgcgg aaccggtatg
     1021 actaatggga tgattctggg cttagatatc ggagtcgcgt ctgtaggggt aggaattatc
     1081 gaggccgata gtggcaaggt aattcacgca agctcacgga tcttccctag tgctaatgcc
     1141 gacaataatg tggagcgccg caagttccgg ggatctaggc gccttcttag gaggaaaaag
     1201 cacagggtta agcgcttaca ggatctgttt gacaagtacg atatcgtgac taacttcgat
     1261 aacctcaacc ttaaccccta cgagctgcga gttaagggct taaacgagcc attgagcaac
     1321 gaagagctct tcgcatcact ccggaacatc acaaagcaca ggggcatttc ctatctcgat
     1381 gatgctgagg atgactcttc tggaaacggg acagaatacg ccaaagcgat agagctgaat
     1441 cagcagcttc tgaaagaaaa gacccccggt cagatccagt acgacagact caatcagtat
     1501 gggcaactga gaggcaattt cgatatcgtg gatgaaaacg gcgagattca ccacgtgata
     1561 aacgtttttt caacatcaag ttacagaaag gaagccgagc agattctcaa gaagcagtct
     1621 gaaacgaata ctagtatcag caccgacttt ataaatgatt tcatccaatt gctgacctct
     1681 aagaggaaat attaccatgg tcctggtaat ccaaagagcc gcacagatta cgggcgctac
     1741 cggacggatg ggacggatct cgataacatc ttcgatgttc tgataggtaa atgcagcttt
     1801 tacccagagg agtaccgagc cagcaagacg agctacactg cccaagagtt caactttctt
     1861 aatgacttga ataacctgac cttaccaacc gagacaggca agttgagcga gcagcagaag
     1921 atcgacctgg tgaattgggc taaggagaca aagatcctcg gaccgaaaaa gctgcttcag
     1981 gaaattgcca agaggaacaa ctgcaagtac gaggacattc gcggctatcg gcttgataag
     2041 aaagataacc ccgatatgca tgtatttgat gtgtatcgga agatgaattt tgacctggag
     2101 actatttccg ttaaggatct gtcagtcgac tctctgaatc agctcgcgcg aattctgaca
     2161 ctgaacaccg agagggaggg gatcgaagag gccatcaaaa atctgatgcc aaaccagttc
     2221 accgagaagc aaatgcttga actcatcgcc ttccgcaaga gtaattcctc tatctttggg
     2281 aaggggtggc acagtctgtc aattaaactg atgaaagagc tgatacccga gctctaccac
     2341 accagtgacg aacaaatgac catactcaat cgatttggta agttcaagct cacgaagctc
     2401 gactcaaaaa ggaccaatta catcgatgaa aactttgtca ctgatgaaat ctataaccct
     2461 gtagtggcca agagtgtgag gcaggcaata aagatcatca acgcttccat taaaaagtgg
     2521 ggggactttg ataagatcgt gattgagatg ccacgcgaca agaatgagga ggaggaaagg
     2581 aaacgaatcg ccgatggcca gaaggtgaat gctaaggaaa aagagcaggc cgagaagcac
     2641 gccgcaaagc tctttaatgg caaggaagag ctcccttctg aagttttcca tggatataag
     2701 gagctggctt tgcgaattag actctggtat cagcaagacc agaagtgcct ctattctggc
     2761 aaggagataa caatttcaga cctgatctac aacagggagc tctttgagat tgaccatatc
     2821 cttccgctgt ctctttcttt tgacgacagt ctgtctaaca aggtcctggt ttacagatgg
     2881 gcaaatcagg agaagggcca gaggacccct ttccaagccc ttgattccat gaaatcagcg
     2941 tggtcctatc gggagttcaa gaatgcaatc ctgcacaatt ctaaaatcag ccggagaaag
     3001 cgtgactatt ttctgacaga acaagacatt agtaagattg aggtgaaaca aaagtttatt
     3061 gagaggaact tggtggacac acggtacgcc agtagaacag ttctcaacgt gctgcagcag
     3121 tccctgaaga atctggagaa ggagactaag gtgtccgttg tccgaggaca gttcacgtcc
     3181 cagctgcgcc ggaaatggca catagataag accagggata cttaccatca ccatgcggtg
     3241 gacgcactga ttatcgcggc ctccgctaag ttgagatatt ggaagaaaca gggcgacatc
     3301 ttgttcgaga actatctcat caatcgccac gtagatagag taaccgggga gatacaatct
     3361 gacgatagct ataaggagga ggtgttcaca cctccctacg acggatttgt ccagactatt
     3421 agcaacccag ggtttgagga cgagatcctt ttctcctatc aggtagacag taaagtcaac
     3481 agaaagatct cagacgccac gatatacgct acgaggtctg cgaagctcga gaaggacaag
     3541 aaggaacaga cgtatgtctt gggtaagata aaagatatct attcacaaac tggttttgag
     3601 aacttcctga agatctataa taaggacaag agtaagttcc tgatctacca gaaggaccct
     3661 gagacttggg aaaagatcat tgaaccaatt ctcaaaaatt atcgggaatt cgataataaa
     3721 ggcaaggata tcgtgaatcc atttgagaaa tacaggaatg ataacgggcc tatctgcaag
     3781 tacagtcgga aaggcaacgg ccctgagatc aaacaattta aatactacga caccgtttac
     3841 aaaattacaa gcggtctcga catcagcccc cgcgaatcaa gaaataaggt aattcttcaa
     3901 agcctgaatc cgtggagaac cgacttctac tttaacccta agactatgaa gtacgaactt
     3961 atgggtatca gatatgtcga cctggagttc gagaaaggaa caggggacta cctgatttct
     4021 gacaatctct ataaagagat taaaaagaac gaggggatct ctgagctgag tgtattcaag
     4081 ttcacactct acaagaacga tctcctgctg atcaaggaca ctgagaacaa cgaagagcaa
     4141 atttttaggt tttggtctcg gaatgacctg tcctccaaaa accgggtgga actgaagccc
     4201 tacgataggt cccgcttttc cggcaatgag atccttatca ccaaaatggg caaggcacct
     4261 aagcaatgca ttaagacttt aacataccaa aacatctcca tttataaaat caaaacagac
     4321 atcctgggat tcaaatacta tctgaaaaac gaaggaaata agccattact gcactttaag
     4381 aagaccggtc ctaagaaaaa gcggaaagtg gctgtttaaa ccgctgatca gcctcgactg
     4441 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4501 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4561 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4621 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     4681 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     4741 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4801 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4861 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4921 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4981 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5041 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5101 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     5161 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     5221 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5281 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     5341 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     5401 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     5461 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     5521 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     5581 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     5641 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     5701 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     5761 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     5821 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     5881 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5941 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     6001 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     6061 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     6121 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     6181 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     6241 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     6301 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     6361 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     6421 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     6481 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     6541 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     6601 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     6661 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     6721 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     6781 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6841 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6901 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6961 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     7021 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     7081 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     7141 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     7201 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     7261 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     7321 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     7381 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     7441 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     7501 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     7561 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     7621 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     7681 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     7741 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     7801 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7861 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7921 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7981 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     8041 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     8101 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     8161 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     8221 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     8281 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     8341 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     8401 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     8461 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     8521 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     8581 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     8641 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     8701 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     8761 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     8821 cgaaaagtgc cacctgacgt c
//
