LOCUS       sequence-238525-        9073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Staphylococcus aureus Cas9 (SaCas9) in Leishmania.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9073)
  AUTHORS   Zhang WW, Matlashewski G
  TITLE     Single-Strand Annealing Plays a Major Role in Double-Strand DNA 
            Break Repair following CRISPR-Cas9 Cleavage in Leishmania.
  JOURNAL   mSphere. 2019 Aug 21;4(4). pii: 4/4/e00408-19. doi: 
            10.1128/mSphere.00408-19.
  PUBMED    31434745
REFERENCE   2  (bases 1 to 9073)
  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: "mSphere."; 
            date: "2019-08-21"; volume: "4(4). pii: 4/4/e00408-19. doi"; pages: 
            " 10.1128/mSphere.00408-19"
COMMENT     Sequence Label: pLPhygSaCas9
FEATURES             Location/Qualifiers
     source          1..9073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..20
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2..19
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1176..1196
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1221..4376
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4377..4424
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(6728..6747)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6729..6747)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6834..6851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7005..7593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7085..7104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7764..8555,8556..8624))
                     /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     8387..8406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8625..8729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8797..8815
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8853..8875)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8975..8994
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gatttaggtg acactataga actcgaccgc tgcttgcgtt cgcatggacg ccacgttcga
       61 cccgatcggg cagcaggccg cggagacgcg gcagcgcaaa cgtgcccggc gggtggctcg
      121 ttccggagga ggcgttccgc cgtcgctacg tcagcatgca agaggatcat tgctttgcgg
      181 ttgaggtggc ggagctcaac taccagcgtg ccgtggaata cgttcatcaa gcgtcagccg
      241 ccggcgcggt cgcgacacct gctcggcgga gcggccactg tgctgctctc ttgacggccg
      301 gtcgcgacgc agggccgctg gtgggtctcc agcgggtacg gtgcgatcag gcagccatga
      361 cggctacgct gagcacgctt gcgaaggagc tggctgcact gtcgccgtag cgcgatgcga
      421 gacgtatcga gcgcatgcca gtggggtgct gggcaacact tggcggcgcg tggctctttc
      481 tcactggttt gggcacgccc acgctgtggg tgtgtcggtg tctgccctgc cgctctctct
      541 cctgatctct cgtcctggca acaacaacac accgcgaatg agttgcccac aagccatctg
      601 cagtccgcac gcgcgtccgg gccttgcggt gtggatgcga gcatcgcgca ttgccggcca
      661 ccgccgcgtg ccaaaagcac acacatcgct ccccctcacg tggtgagcgt ctgcgcgtgt
      721 gtgtctgtgt gcataaatgc gtacgtgtgc gctcggtcca cggtcaccat cgcgccccgc
      781 ggcatcggtg ctcgcacctt tccattctct ccgtacggcg ttcgccactc tgctaactct
      841 tctccccttt cctctctccg catctgcgcc ttgtgtgccg tgtgtgcgcc ccacacgtac
      901 tctacacgcc ttctctgcat gcgctcctcg cctgcctgaa acgatgacgt gcgccgccac
      961 tcgctcaccc gctcccatcc acatgcattc ctgcacccta cagcgactct aagccgtaca
     1021 ttccatctcc tcaacaccca ctcgcttcct tctgctctca cctattactt cgccagccca
     1081 catatctgct ataacctgcc ctcccccacc cgcttcccac acatccgcca ccgctacgaa
     1141 gcctcgactc tagaccatgg aagcttacca tggccccaaa gaagaagcgg aaggtcggta
     1201 tccacggagt cccagcagcc aagcggaact acatcctggg cctggacatc ggcatcacca
     1261 gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc
     1321 tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa
     1381 ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca
     1441 acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg
     1501 gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga
     1561 gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca
     1621 aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc
     1681 tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg
     1741 actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc
     1801 agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac
     1861 ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg
     1921 gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc
     1981 tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc
     2041 tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca
     2101 ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag
     2161 tgaccagcac cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca
     2221 ttaccgcccg gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc
     2281 tgaccatcta ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc
     2341 tgacccagga agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc
     2401 tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga
     2461 tcgctatctt caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag
     2521 agatccccac caccctggtg gacgacttca tcctgagccc cgtcgtgaag aggagcttca
     2581 tccagagcat caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca
     2641 ttatcgagct ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc
     2701 agaagcggaa ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag
     2761 agaacgccaa gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc
     2821 tgtacagcct ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg
     2881 tggaccacat catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg
     2941 tgaagcagga agaaaacagc aagaagggca accggacccc attccagtac ctgagcagca
     3001 gcgacagcaa gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca
     3061 agggcagaat cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt
     3121 tctccgtgca gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag
     3181 gcctgatgaa cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt
     3241 ccatcaatgg cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga
     3301 acaaggggta caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct
     3361 tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg
     3421 aaaagcaggc cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca
     3481 tcacccccca ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg
     3541 tggacaagaa gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg
     3601 acaagggcaa caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca
     3661 agctgaaaaa gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc
     3721 agacctacca gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt
     3781 acaagtacta cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc
     3841 ccgtgatcaa gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg
     3901 acgactaccc caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg
     3961 acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca
     4021 aaaaagaaaa ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga
     4081 agatcagcaa ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca
     4141 acggcgagct gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga
     4201 acatgatcga catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca
     4261 ggatcattaa gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc
     4321 tgggcaacct gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa
     4381 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaggat ccgcggcccc
     4441 gcctcgagcc cgggcgccgt cgaccgctgc ttgcgttcgc atggacgcca cgttcgaccc
     4501 gatcgggcag caggccgcgg agacgcggca gcgcaaacgt gcccggcggg tggctcgttc
     4561 cggaggaggc gttccgccgt cgctacgtca gcatgcaaga ggatcattgc tttgcggttg
     4621 aggtggcgga gctcaactac cagcgtgccg tggaatacgt tcatcaagcg tcagccgccg
     4681 gcgcggtcgc gacacctgct cggcggagcg gccactgtgc tgctctcttg acggccggtc
     4741 gcgacgcagg gccgctggtg ggtctccagc gggtacggtg cgatcaggca gccatgacgg
     4801 ctacgctgag cacgcttgcg aaggagctgg ctgcactgtc gccgtagcgc gatgcgagac
     4861 gtatcgagcg catgccagtg gggtgctggg caacacttgg cggcgcgtgg ctctttctca
     4921 ctggtttggg cacgcccacg ctgtgggtgt gtcggtgtct gccctgccgc tctctctcct
     4981 gatctctcgt cctggcaaca acaacacacc gcgaatgagt tgcccacaag ccatctgcag
     5041 tccgcacgcg cgtccgggcc ttgcggtgtg gatgcgagca tcgcgcattg ccggccaccg
     5101 ccgcgtgcca aaagcacaca catcgctccc cctcacgtgg tgagcgtctg cgcgtgtgtg
     5161 tctgtgtgca taaatgcgta cgtgtgcgct cggtccacgg tcaccatcgc gccccgcggc
     5221 atcggtgctc gcacctttcc attctctccg tacggcgttc gccactctgc taactcttct
     5281 cccctttcct ctctccgcat ctgcgccttg tgtgccgtgt gtgcgcccca cacgtactct
     5341 acacgccttc tctgcatgcg ctcctcgcct gcctgaaacg atgacgtgcg ccgccactcg
     5401 ctcacccgct cccatccaca tgcattcctg caccctacag cgactctaag ccgtacattc
     5461 catctcctca acacccactc gcttccttct gctctcacct attacttcgc cagcccacat
     5521 atctgctata acctgccctc ccccacccgc ttcccacaca tccgccaccg ctacgaagcc
     5581 tcggtaccga gctctctctt ctccttcccc tctctctcct ctctcctctc tccagagctc
     5641 tctcttctcc ttcccctctc tctcctctct cctctctcca gagctcgaat tgatatgaaa
     5701 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     5761 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     5821 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     5881 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     5941 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     6001 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     6061 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     6121 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     6181 caaactgtta tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     6241 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     6301 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     6361 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     6421 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     6481 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     6541 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     6601 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     6661 gaagtactcg ccgatagtgg aaacagacgc cccagcactc gtccgggatc atcagatctg
     6721 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga
     6781 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6841 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6901 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6961 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     7021 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     7081 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7141 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     7201 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7261 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7321 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7381 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7441 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7501 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7561 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7621 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7681 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7741 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7801 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7861 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7921 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7981 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     8041 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     8101 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     8161 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     8221 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     8281 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8341 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     8401 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8461 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8521 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8581 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8641 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8701 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     8761 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     8821 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     8881 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     8941 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     9001 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt
     9061 atcatacaca tac
//
