LOCUS       sequence-118367-        3677 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 3677)
  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 3677)
  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: pSPneogRNAH
FEATURES             Location/Qualifiers
     source          1..3677
                     /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"
     misc_RNA        237..312
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           319..386
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     CDS             508..1302
                     /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
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(562..581)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1172..1191
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1332..1351)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1333..1351)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1438..1455)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1609..2197)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1689..1708)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2368..3159,3160..3228))
                     /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     2991..3010
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3229..3333)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3401..3419
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3457..3479)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3579..3598
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gatttaggtg acactataga actcgagcag ctgaagcttg tgagttatga ggtctgcgat
       61 tgacgtagga gttgcaaggg ggagggggta tgaacggggt gggtagagct tttttaggtg
      121 gaagtagtga gagggtgggc ttgtgagaat ttgaggtgtg ttcgtgatgt gtggatctta
      181 tcgggggctc ggttgagttt ttggtttggt gattttgtgg gtcttcgaga agacctgttt
      241 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      301 ccgagtcggt gcttttttgg ccggcatggt cccagcctcc tcgctggcgc cggctgggca
      361 acatgcttcg gcatggcgaa tgggacggat ccccgggtac cgagctctct cttctccttc
      421 ccctctctct cctctctcct ctctccagag ctctctcttc tccttcccct ctctctcctc
      481 tctcctctct ccagagctcg aattcgcatg attgaacaag atggattgca cgcaggttct
      541 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
      601 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
      661 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
      721 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
      781 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
      841 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
      901 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
      961 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     1021 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     1081 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     1141 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     1201 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     1261 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gatcgatgat atcagatcga
     1321 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     1381 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     1441 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1501 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1561 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1621 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1681 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1741 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1801 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1861 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1921 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1981 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2041 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2101 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2161 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2221 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2281 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2341 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2401 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2461 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2521 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2581 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2641 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2701 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2761 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2821 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2881 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2941 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3001 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3061 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3121 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3181 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3241 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3301 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3361 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3421 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3481 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3541 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     3601 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt
     3661 atgtatcata cacatac
//
