LOCUS       sequence-238734-        4843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA of Staphylococcus aureus Cas9 (SaCas9) in Leishmania.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4843)
  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 4843)
  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: pNeoSagRNAH
FEATURES             Location/Qualifiers
     source          1..4843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        225..300
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     ncRNA           306..373
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     CDS             1686..2480
                     /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(1740..1759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2350..2369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2497..2516)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2498..2516)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2603..2620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2774..3362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2854..2873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3533..4324,4325..4393))
                     /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     4156..4175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4394..4498)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4566..4584
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4622..4644)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4744..4763
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa ctcgagtgtg agttatgagg tctgcgattg acgtaggagt
       61 tgcaaggggg agggggtatg aacggggtgg gtagagcttt tttaggtgga agtagtgaga
      121 gggtgggctt gtgagaattt gaggtgtgtt cgtgatgtgt ggatcttatc gggggctcgg
      181 ttgagttttt ggtttggtga ttttgtgggt cttcgagaag acctgtttta gtactctgga
      241 aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga
      301 tttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca
      361 tggcgaatgg gactgatccc gggtaccgag ctctctcttc tccttcccct ctctctcctc
      421 tctcctctct ccagagctct ctcttctcct tcccctctct ctcctctctc ctctctccag
      481 agctcgaatt cgcaagcttg catgcctgca gggatccgcg gccgcctcga gcccgggcgc
      541 cgtcgaccgc tgcttgcgtt cgcatggacg ccacgttcga cccgatcggg cagcaggccg
      601 cggagacgcg gcagcgcaaa cgtgcccggc gggtggctcg ttccggagga ggcgttccgc
      661 cgtcgctacg tcagcatgca agaggatcat tgctttgcgg ttgaggtggc ggagctcaac
      721 taccagcgtg ccgtggaata cgttcatcaa gcgtcagccg ccggcgcggt cgcgacacct
      781 gctcggcgga gcggccactg tgctgctctc ttgacggccg gtcgcgacgc agggccgctg
      841 gtgggtctcc agcgggtacg gtgcgatcag gcagccatga cggctacgct gagcacgctt
      901 gcgaaggagc tggctgcact gtcgccgtag cgcgatgcga gacgtatcga gcgcatgcca
      961 gtggggtgct gggcaacact tggcggcgcg tggctctttc tcactggttt gggcacgccc
     1021 acgctgtggg tgtgtcggtg tctgccctgc cgctctctct cctgatctct cgtcctggca
     1081 acaacaacac accgcgaatg agttgcccac aagccatctg cagtccgcac gcgcgtccgg
     1141 gccttgcggt gtggatgcga gcatcgcgca ttgccggcca ccgccgcgtg ccaaaagcac
     1201 acacatcgct ccccctcacg tggtgagcgt ctgcgcgtgt gtgtctgtgt gcataaatgc
     1261 gtacgtgtgc gctcggtcca cggtcaccat cgcgccccgc ggcatcggtg ctcgcacctt
     1321 tccattctct ccgtacggcg ttcgccactc tgctaactct tctccccttt cctctctccg
     1381 catctgcgcc ttgtgtgccg tgtgtgcgcc ccacacgtac tctacacgcc ttctctgcat
     1441 gcgctcctcg cctgcctgaa acgatgacgt gcgccgccac tcgctcaccc gctcccatcc
     1501 acatgcattc ctgcacccta cagcgactct aagccgtaca ttccatctcc tcaacaccca
     1561 ctcgcttcct tctgctctca cctattactt cgccagccca catatctgct ataacctgcc
     1621 ctcccccacc cgcttcccac acatccgcca ccgctacgaa gcctcggtac cgagctcgaa
     1681 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     1741 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     1801 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     1861 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     1921 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     1981 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     2041 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac aaagcgaaac
     2101 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     2161 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     2221 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     2281 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     2341 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     2401 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     2461 ttcttgacga gttcttctga tcggatctgc cggtctccct atagtgagtc gtattaattt
     2521 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2581 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2641 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2701 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2761 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2821 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2881 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2941 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3001 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3061 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3121 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3181 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3241 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3301 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3361 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3421 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3481 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3541 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3601 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3661 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3721 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3781 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3841 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3901 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3961 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4021 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4081 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4141 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4201 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4261 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4321 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4381 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4501 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4561 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     4621 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4681 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     4741 atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac
     4801 gcggctacaa ttaatacata accttatgta tcatacacat acg
//
