LOCUS       sequence-238733-        8091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Staphylococcus aureus Cas9 (SaCas9) and its gRNA in 
            Leishmania.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8091)
  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 8091)
  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: pLdSaCN
FEATURES             Location/Qualifiers
     source          1..8091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        206..281
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     ncRNA           287..354
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     CDS             490..510
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             535..3690
                     /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             3691..3738
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4915..5709
                     /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
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHKAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4969..4988)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5579..5598
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5726..5745)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5727..5745)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5832..5849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6003..6591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6083..6102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6762..7553,7554..7622))
                     /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     7385..7404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7623..7727)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7795..7813
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7851..7873)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7973..7992
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        8073..8091
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     8073..8090
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 actcgagtgt gagttatgag gtctgcgatt gacgtaggag ttgcaagggg gagggggtat
       61 gaacggggtg ggtagagctt ttttaggtgg aagtagtgag agggtgggct tgtgagaatt
      121 tgaggtgtgt tcgtgatgtg tggatcttat cgggggctcg gttgagtttt tggtttggtg
      181 attttgtggg tcttcgagaa gacctgtttt agtactctgg aaacagaatc tactaaaaca
      241 aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag atttttggcc ggcatggtcc
      301 cagcctcctc gctggcgccg gctgggcaac atgcttcggc atggcgaatg ggactgatcc
      361 cgggtaccga gctctctctt ctccttcccc tctctctcct ctctcctctc tccagagctc
      421 tctcttctcc ttcccctctc tctcctctct cctctctcca gagctcgaat tcgcaagctt
      481 accatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccaagcgg
      541 aactacatcc tgggcctgga catcggcatc accagcgtgg gctacggcat catcgactac
      601 gagacacggg acgtgatcga tgccggcgtg cggctgttca aagaggccaa cgtggaaaac
      661 aacgagggca ggcggagcaa gagaggcgcc agaaggctga agcggcggag gcggcataga
      721 atccagagag tgaagaagct gctgttcgac tacaacctgc tgaccgacca cagcgagctg
      781 agcggcatca acccctacga ggccagagtg aagggcctga gccagaagct gagcgaggaa
      841 gagttctctg ccgccctgct gcacctggcc aagagaagag gcgtgcacaa cgtgaacgag
      901 gtggaagagg acaccggcaa cgagctgtcc accaaagagc agatcagccg gaacagcaag
      961 gccctggaag agaaatacgt ggccgaactg cagctggaac ggctgaagaa agacggcgaa
     1021 gtgcggggca gcatcaacag attcaagacc agcgactacg tgaaagaagc caaacagctg
     1081 ctgaaggtgc agaaggccta ccaccagctg gaccagagct tcatcgacac ctacatcgac
     1141 ctgctggaaa cccggcggac ctactatgag ggacctggcg agggcagccc cttcggctgg
     1201 aaggacatca aagaatggta cgagatgctg atgggccact gcacctactt ccccgaggaa
     1261 ctgcggagcg tgaagtacgc ctacaacgcc gacctgtaca acgccctgaa cgacctgaac
     1321 aatctcgtga tcaccaggga cgagaacgag aagctggaat attacgagaa gttccagatc
     1381 atcgagaacg tgttcaagca gaagaagaag cccaccctga agcagatcgc caaagaaatc
     1441 ctcgtgaacg aagaggatat taagggctac agagtgacca gcaccggcaa gcccgagttc
     1501 accaacctga aggtgtacca cgacatcaag gacattaccg cccggaaaga gattattgag
     1561 aacgccgagc tgctggatca gattgccaag atcctgacca tctaccagag cagcgaggac
     1621 atccaggaag aactgaccaa tctgaactcc gagctgaccc aggaagagat cgagcagatc
     1681 tctaatctga agggctatac cggcacccac aacctgagcc tgaaggccat caacctgatc
     1741 ctggacgagc tgtggcacac caacgacaac cagatcgcta tcttcaaccg gctgaagctg
     1801 gtgcccaaga aggtggacct gtcccagcag aaagagatcc ccaccaccct ggtggacgac
     1861 ttcatcctga gccccgtcgt gaagaggagc ttcatccaga gcatcaaagt gatcaacgcc
     1921 atcatcaaga agtacggcct gcccaacgac atcattatcg agctggcccg cgagaagaac
     1981 tccaaggacg cccagaaaat gatcaacgag atgcagaagc ggaaccggca gaccaacgag
     2041 cggatcgagg aaatcatccg gaccaccggc aaagagaacg ccaagtacct gatcgagaag
     2101 atcaagctgc acgacatgca ggaaggcaag tgcctgtaca gcctggaagc catccctctg
     2161 gaagatctgc tgaacaaccc cttcaactat gaggtggacc acatcatccc cagaagcgtg
     2221 tccttcgaca acagcttcaa caacaaggtg ctcgtgaagc aggaagaaaa cagcaagaag
     2281 ggcaaccgga ccccattcca gtacctgagc agcagcgaca gcaagatcag ctacgaaacc
     2341 ttcaagaagc acatcctgaa tctggccaag ggcaagggca gaatcagcaa gaccaagaaa
     2401 gagtatctgc tggaagaacg ggacatcaac aggttctccg tgcagaaaga cttcatcaac
     2461 cggaacctgg tggataccag atacgccacc agaggcctga tgaacctgct gcggagctac
     2521 ttcagagtga acaacctgga cgtgaaagtg aagtccatca atggcggctt caccagcttt
     2581 ctgcggcgga agtggaagtt taagaaagag cggaacaagg ggtacaagca ccacgccgag
     2641 gacgccctga tcattgccaa cgccgatttc atcttcaaag agtggaagaa actggacaag
     2701 gccaaaaaag tgatggaaaa ccagatgttc gaggaaaagc aggccgagag catgcccgag
     2761 atcgaaaccg agcaggagta caaagagatc ttcatcaccc cccaccagat caagcacatt
     2821 aaggacttca aggactacaa gtacagccac cgggtggaca agaagcctaa tagagagctg
     2881 attaacgaca ccctgtactc cacccggaag gacgacaagg gcaacaccct gatcgtgaac
     2941 aatctgaacg gcctgtacga caaggacaat gacaagctga aaaagctgat caacaagagc
     3001 cccgaaaagc tgctgatgta ccaccacgac ccccagacct accagaaact gaagctgatt
     3061 atggaacagt acggcgacga gaagaatccc ctgtacaagt actacgagga aaccgggaac
     3121 tacctgacca agtactccaa aaaggacaac ggccccgtga tcaagaagat taagtattac
     3181 ggcaacaaac tgaacgccca tctggacatc accgacgact accccaacag cagaaacaag
     3241 gtcgtgaagc tgtccctgaa gccctacaga ttcgacgtgt acctggacaa tggcgtgtac
     3301 aagttcgtga ccgtgaagaa tctggatgtg atcaaaaaag aaaactacta cgaagtgaat
     3361 agcaagtgct atgaggaagc taagaagctg aagaagatca gcaaccaggc cgagtttatc
     3421 gcctccttct acaacaacga tctgatcaag atcaacggcg agctgtatag agtgatcggc
     3481 gtgaacaacg acctgctgaa ccggatcgaa gtgaacatga tcgacatcac ctaccgcgag
     3541 tacctggaaa acatgaacga caagaggccc cccaggatca ttaagacaat cgcctccaag
     3601 acccagagca ttaagaagta cagcacagac attctgggca acctgtatga agtgaaatct
     3661 aagaagcacc ctcagatcat caaaaagggc aaaaggccgg cggccacgaa aaaggccggc
     3721 caggcaaaaa agaaaaagta ggatccgcgg ccgcctcgag cccgggcgcc gtcgaccgct
     3781 gcttgcgttc gcatggacgc cacgttcgac ccgatcgggc agcaggccgc ggagacgcgg
     3841 cagcgcaaac gtgcccggcg ggtggctcgt tccggaggag gcgttccgcc gtcgctacgt
     3901 cagcatgcaa gaggatcatt gctttgcggt tgaggtggcg gagctcaact accagcgtgc
     3961 cgtggaatac gttcatcaag cgtcagccgc cggcgcggtc gcgacacctg ctcggcggag
     4021 cggccactgt gctgctctct tgacggccgg tcgcgacgca gggccgctgg tgggtctcca
     4081 gcgggtacgg tgcgatcagg cagccatgac ggctacgctg agcacgcttg cgaaggagct
     4141 ggctgcactg tcgccgtagc gcgatgcgag acgtatcgag cgcatgccag tggggtgctg
     4201 ggcaacactt ggcggcgcgt ggctctttct cactggtttg ggcacgccca cgctgtgggt
     4261 gtgtcggtgt ctgccctgcc gctctctctc ctgatctctc gtcctggcaa caacaacaca
     4321 ccgcgaatga gttgcccaca agccatctgc agtccgcacg cgcgtccggg ccttgcggtg
     4381 tggatgcgag catcgcgcat tgccggccac cgccgcgtgc caaaagcaca cacatcgctc
     4441 cccctcacgt ggtgagcgtc tgcgcgtgtg tgtctgtgtg cataaatgcg tacgtgtgcg
     4501 ctcggtccac ggtcaccatc gcgccccgcg gcatcggtgc tcgcaccttt ccattctctc
     4561 cgtacggcgt tcgccactct gctaactctt ctcccctttc ctctctccgc atctgcgcct
     4621 tgtgtgccgt gtgtgcgccc cacacgtact ctacacgcct tctctgcatg cgctcctcgc
     4681 ctgcctgaaa cgatgacgtg cgccgccact cgctcacccg ctcccatcca catgcattcc
     4741 tgcaccctac agcgactcta agccgtacat tccatctcct caacacccac tcgcttcctt
     4801 ctgctctcac ctattacttc gccagcccac atatctgcta taacctgccc tcccccaccc
     4861 gcttcccaca catccgccac cgctacgaag cctcggtacc gagctcgaat tcgcatgatt
     4921 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4981 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     5041 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     5101 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     5161 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5221 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5281 ctgcatacgc ttgatccggc tacctgccca ttcgaccaca aagcgaaaca tcgcatcgag
     5341 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5401 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5461 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5521 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5581 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5641 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5701 ttcttctgat cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     5761 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5821 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5881 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5941 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6001 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6061 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6121 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6181 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6241 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6301 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6361 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6421 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6481 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6541 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6601 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6661 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6721 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6781 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6841 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6901 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6961 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7021 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7081 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7141 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7201 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7261 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7321 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7381 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7441 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7501 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7561 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7621 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7681 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7741 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     7801 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     7861 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     7921 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     7981 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     8041 taatacataa ccttatgtat catacacata cgatttaggt gacactatag a
//
