LOCUS       sequence-170932-        3742 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA in Leishmania.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3742)
  AUTHORS   Zhang WW, Matlashewski G
  TITLE     CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.
  JOURNAL   MBio. 2015 Jul 21;6(4). pii: e00861-15. doi: 10.1128/mBio.00861-15.
  PUBMED    26199327
REFERENCE   2  (bases 1 to 3742)
  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: "MBio."; 
            date: "2015-07-21"; volume: "6(4). pii: e00861-15. doi"; pages: " 
            10.1128/mBio.00861-15"
COMMENT     Sequence Label: pSPneoHHgRNAH
FEATURES             Location/Qualifiers
     source          1..3742
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..52)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(206..794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(286..305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(965..1756,1757..1825))
                     /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     1588..1607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1826..1930)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1998..2016
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2054..2076)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2176..2195
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2276..2294
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2276..2293
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     ncRNA           2515..2551
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        2576..2651
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           2658..2725
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     CDS             2847..3641
                     /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(2901..2920)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3511..3530
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3671..3690)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3672..3690)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
       61 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      121 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      181 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      241 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      301 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      361 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      421 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      481 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      541 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      601 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
      661 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      721 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      781 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      841 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
      901 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
      961 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1021 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1081 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1141 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1201 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1261 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1321 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     1381 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     1441 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     1501 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     1561 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     1621 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     1681 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     1741 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     1801 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     1861 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1921 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     1981 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     2041 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     2101 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     2161 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat
     2221 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta
     2281 ggtgacacta tagaactcga gcagctgaag cttgtgagtt atgaggtctg cgattgacgt
     2341 aggagttgca agggggaggg ggtatgaacg gggtgggtag agctttttta ggtggaagta
     2401 gtgagagggt gggcttgtga gaatttgagg tgtgttcgtg atgtgtggat cttatcgggg
     2461 gctcggttga gtttttggtt tggtgatttt gtaggaagct taagatctca ccatctgatg
     2521 agtccgtgag gacgaaacga gtaagctcgt catggtgggg tcttcgagaa gacctgtttt
     2581 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2641 cgagtcggtg cttttttggc cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa
     2701 catgcttcgg catggcgaat gggacggatc cccgggtacc gagctctctc ttctccttcc
     2761 cctctctctc ctctctcctc tctccagagc tctctcttct ccttcccctc tctctcctct
     2821 ctcctctctc cagagctcga attcgcatga ttgaacaaga tggattgcac gcaggttctc
     2881 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2941 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3001 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3061 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3121 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3181 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3241 cattcgacca caaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3301 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3361 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3421 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3481 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3541 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3601 agcgcatcgc cttctatcgc cttcttgacg agttcttctg atcgatgata tcagatcgat
     3661 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa
     3721 tgaatcggcc aacgcgcggg ga
//
