LOCUS       sequence-238735-        3746 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 3746)
  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 3746)
  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: pSPneoSagRNAH
FEATURES             Location/Qualifiers
     source          1..3746
                     /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             580..1374
                     /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(634..653)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1244..1263
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1400..1419)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1401..1419)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1506..1523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1677..2265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1757..1776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2436..3227,3228..3296))
                     /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     3059..3078
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3297..3401)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3469..3487
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3525..3547)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3647..3666
                     /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 gggtaatgaa atcggctgga ctttgcttga ttttctgctc
      421 gaattcgcaa gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgagctct
      481 ctcttctcct tcccctctct ctcctctctc ctctctccag agctctctct tctccttccc
      541 ctctctctcc tctctcctct ctccagagct cgaattcgca tgattgaaca agatggattg
      601 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
      661 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
      721 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
      781 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
      841 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
      901 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
      961 ccggctacct gcccattcga ccacaaagcg aaacatcgca tcgagcgagc acgtactcgg
     1021 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     1081 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     1141 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     1201 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     1261 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     1321 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgatcgatg
     1381 atatcagatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
     1441 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1501 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1561 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1621 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1681 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1741 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1801 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1861 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1921 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1981 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2041 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2101 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2161 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2221 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2281 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2341 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2401 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2461 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2521 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2581 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2641 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2701 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2761 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2821 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2881 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2941 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3001 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3061 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3121 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3181 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3241 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3301 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3361 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3421 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     3481 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     3541 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     3601 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     3661 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     3721 ataaccttat gtatcataca catacg
//
