LOCUS       sequence-490412-        4491 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  Backbone for cloning sgRNAs to be used with CRISPR Cas9 cytosine 
            base editor. Expressed from the 18S rRNA SSU locus in Leishmania. 
            Contains puromycin selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4491)
  AUTHORS   Herrmann May N, Cao A, Schmid A, Link F, Arias-Del-Angel J, Meiser 
            E, Beneke T
  TITLE     Improved base editing and functional screening in Leishmania via 
            co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, 
            and a cytosine base editor.
  JOURNAL   eLife. 2025 Feb 24;13:RP97437. doi: 10.7554/eLife.97437.
  PUBMED    39991929
REFERENCE   2  (bases 1 to 4491)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eLife."; 
            date: "2025-02-24"; volume: "13:RP97437. doi"; pages: " 
            10.7554/eLife.97437"
COMMENT     Sequence Label: pTB105
FEATURES             Location/Qualifiers
     source          1..4491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      171..759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     660..679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        2163..2238
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           2245..2312
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     rep_origin      2789..3244
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2876..2895)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3086..3107
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3261..3280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3380..3402
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3440..3458)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3526..3630
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3631..3699,3700..4491)
                     /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     complement(3849..3868)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
       61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      301 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      781 ttacggttcc tggccttttg ctggcctttt gctcacatgt aagcttttaa ttaagaccgc
      841 accaagacga actacagcga aggcattctt caaggatacc ttcctcaatc aagaaccaaa
      901 gtgtggagat cgaagatgat tagagaccat tgtagtccac actgcaaacg atgacaccca
      961 tgaattgggg atcttatggg ccggcctgcg gcagggttta ccctgtgtca gcaccgcgcc
     1021 cgcttttacc aacttacgta tcttttctat tcggccttta ccggccaccc acgggaatat
     1081 cctcagcacg ttttctgttt tttcacgcga aagctttgag gttacagtct caggggtgag
     1141 tacgttcgca agagtgaaac ttaaagaaat tgacggaatg gcaccacaag acgtggagcg
     1201 tgcgggtata atgcagacct gctgcaagct aggggcacag caaggtgatc tgaaattcat
     1261 gttttttttt ttttactctg cattgcagtc tccgctctta tttagttttg ctttacgtaa
     1321 ggtctcgttg ctgccataaa ataagctcta gtggaattca tgactgaata caagccaacg
     1381 gttcgtcttg ctacgcgaga cgatgtgccg cgggccgtac ggacgctggc tgctgcgttc
     1441 gctgattatc ccgccacccg acatacggtt gacccagatc gacacattga gcgagtcaca
     1501 gaacttcaag agttattctt aacacgggtg ggcttggaca ttggtaaggt ttgggttgcg
     1561 gacgatggcg cggctgttgc ggtgtggacg acgccagagt cagtcgaagc gggcgcagtg
     1621 tttgctgaga tcggtccccg aatggcggag cttagcgggt catgcctcgc tgcacaacag
     1681 cagatggaag gtctgttggc accacataga cctaaggagc cggcttggtt cttagcgaca
     1741 gttggcgtca gtccggacca ccaaggtaag gggctgggtt cagcggttgt tcttccagga
     1801 gtagaggctg cggaaagggc aggtgtgccg gcgttccttg aaacgtctgc accacgtaac
     1861 cttccttttt acgagagatt ggggtttaca gtaacagcag atgttgaagt gcctgaaggg
     1921 ccgaggacat ggtgcatgac acgtaagccg ggagcctaac catggtgccc atttagttgg
     1981 cctttccctt gtctcgtgtc ttttccgtgg aaaggttccc ggagtaatct gatggcacag
     2041 cagggaggtg cgcctgcagg ttggttagga aggggggatg atgtaaaaga agaaaatggg
     2101 gggattcgag ccccacgcgt gaaattaata cgtctcacta tagggggtct tcgagaagac
     2161 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     2221 gtggcaccga gtcggtgctt ttttggccgg catggtccca gcctcctcgc tggcgccggc
     2281 tgggcaacat gcttcggcat ggcgaatggg acgtgctgtg ctttttcctt tatcgtgtca
     2341 tgcatagctt gagtattcct ttaccagatc cggacaggat gaggattgac agattgagtg
     2401 ttctttctcg attccctgaa tggtggtgca tggccgcttt tggtcggtgg agtgatttgt
     2461 ttggttgatt ccgtcaacgg acgagatcca agctgcccag tagaattcag aattgcccat
     2521 aggatagcaa actcatcggc gggttttacc caacggtggg ccgcattcgg tcgaattctt
     2581 ctctgcggga ttcctttgta attgcacaag gtgaaatttt gggcaacagc aggtctgtga
     2641 tgctcctcaa tgttctgggc gacacgcgca ctacaatgtc agtgagaact taattaaaat
     2701 ccctagcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2761 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     2821 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     2881 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2941 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3001 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3061 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3121 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     3181 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3241 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3301 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3361 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3421 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3481 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3541 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3601 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3661 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3721 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3781 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3841 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3901 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3961 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4021 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4081 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4141 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4201 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4261 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4321 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     4381 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4441 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta a
//
