LOCUS       sequence-160871-        3900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express  LdPBK_241510.1 and LdMT targeting  gRNAs simutaneously.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3900)
  AUTHORS   Zhang WW, Lypaczewski P, Matlashewski G
  TITLE     Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To 
            Target a Multigene Family, Induce Chromosomal Translocation, and 
            Study DNA Break Repair Mechanisms.
  JOURNAL   mSphere. 2017 Jan 18;2(1). pii: e00340-16. doi: 
            10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb.
  PUBMED    28124028
REFERENCE   2  (bases 1 to 3900)
  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: "2017-01-18"; volume: "2(1). pii: e00340-16. doi"; pages: " 
            10.1128/mSphere.00340-16. eCollection 2017 Jan-Feb"
COMMENT     Sequence Label: pSPneogRNA241510+MT
FEATURES             Location/Qualifiers
     source          1..3900
                     /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"
     ncRNA           399..435
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        461..536
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           543..610
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     CDS             731..1525
                     /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(785..804)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1395..1414
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1555..1574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1556..1574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1661..1678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1832..2420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1912..1931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2591..3382,3383..3451))
                     /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     3214..3233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3452..3556)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3624..3642
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3680..3702)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3802..3821
                     /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 ttgagagaat ttgaggtgtg ttcgtgatgt gtggatctta
      181 tcgggggctc ggttgagttt ttggtttggt gattttgtgg tgtcgttctg cagccagttt
      241 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      301 ccgagtcggt gcttttttgg ccggcatggt cccagcctcc tcgctggcgc cggctgggca
      361 acatgcttcg gcatggcgaa tgggacggat ctcaccatct gatgagtccg tgaggacgaa
      421 acgagtaagc tcgtcatggt ggtccaggcg cagtggatgc gttttagagc tagaaatagc
      481 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      541 ttggccggca tggtcccagc ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg
      601 cgaatgggac ggatcccggg taccgagctc tctcttctcc ttcccctctc tctcctctct
      661 cctctctcca gagctctctc ttctccttcc cctctctctc ctctctcctc tctccagagc
      721 tcgaattcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
      781 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
      841 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
      901 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
      961 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     1021 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     1081 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     1141 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     1201 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     1261 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     1321 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     1381 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     1441 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     1501 tcgccttctt gacgagttct tctgatcgat gatatcagat cgatctgccg gtctccctat
     1561 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     1621 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1681 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1741 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1801 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1861 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1921 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1981 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     2041 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2101 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2161 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2221 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     2281 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2341 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2401 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2461 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2521 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2581 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2641 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2701 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2761 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2821 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2881 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2941 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3001 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3061 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3121 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3181 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3241 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3301 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3361 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3421 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3481 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3541 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     3601 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     3661 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3721 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3781 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     3841 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
//
