LOCUS       sequence-212484-        4431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA in T. brucei cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4431)
  AUTHORS   Rico E, Jeacock L, Kovarova J, Horn D
  TITLE     Inducible high-efficiency CRISPR-Cas9-targeted gene editing and 
            precision base editing in African trypanosomes.
  JOURNAL   Sci Rep. 2018 May 21;8(1):7960. doi: 10.1038/s41598-018-26303-w.
  PUBMED    29785042
REFERENCE   2  (bases 1 to 4431)
  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: "Sci Rep."; 
            date: "2018-05-21"; volume: "8(1):7960. doi"; pages: " 
            10.1038/s41598-018-26303-w"
COMMENT     Sequence Label: pT7sgRNA
FEATURES             Location/Qualifiers
     source          1..4431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(657..674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(828..1416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(908..927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1587..2378,2379..2447))
                     /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     2210..2229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2448..2552)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2620..2638
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2676..2698)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2798..2817
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3027..3046
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(3411..3785)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     4226..4245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4226..4244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        4264..4339
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           4346..4413
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
ORIGIN
        1 gaattcgatg cgaggcgaat cgctcagtag tgttagtgca ggcattgttt ctcttttcca
       61 tttcggctcg ctctctgtgc cagaaaggct tttggagtct tggttactcg tgggactagg
      121 gcaagcgtcg cttccctaat aattgttgct aacgtttcac cgtgcgtgtg tggatatctt
      181 ccaagttgtc attttctcta cccctctctt ttccggcgcg gtctaacttc ctccaactag
      241 cgcatttcca tgataacggg gtccacatag tccgttgtag tgaaattggc atgccatcag
      301 ggccaaggca tacatcgcgg ccgcgataaa aatgatacga tgatctccgt gtggagcaga
      361 ccgtggcgac cagtttgtac catggaagtt cttcgataga gttatatcga gtgcggaatt
      421 gatgctacgg tcacagtgtc gtatcgtgtg gtctcccatg tgtttttgac ggtgtaccaa
      481 ttatctttgc actaatgggc tatggaatct ttccttttaa tcagggggac atgtttatcc
      541 cacgccagta gtctggaatt agaatacata ccaactatat gagctcgaat tctgcattaa
      601 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
      661 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      721 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      781 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      841 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      901 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      961 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1021 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1081 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1141 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1201 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1261 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1321 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1381 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1441 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1501 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1561 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1621 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1681 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1741 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1801 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1861 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1921 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1981 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2041 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2101 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2161 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2221 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2281 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2341 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2401 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2461 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2521 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     2581 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     2641 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     2701 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     2761 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     2821 agagtgcacc attcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     2881 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcaaggagat ggcgcccaac
     2941 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     3001 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     3061 cctgtggcgc cggtgatgcc ggtctgcata gataacaaac gcatcaaaca caactggtgg
     3121 cactatttca atcatgtcga cacaccaaga cgagaaaatt tgagttaaac aagatgccta
     3181 caaccctata ttattatctt caagtgatct tctcttctct tgtgcgctgt acgtaaatgt
     3241 gttgcaaata aggcataggg ctgagtacag gcaccacacg cgaggaaaat cataagcact
     3301 agccgtgtca actcacagcg actacagaac aattttggcc acacaacccg gtgttaggat
     3361 ccctcggacc cgtcgggccg cgtcggaccg gcggtgttgg tcggcgtcgg tcagtcctgc
     3421 tcctcggcca cgaagtgcac gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc
     3481 cacggctgct cgccgatctc ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac
     3541 acgacctccg accactcggc gtacagctcg tccaggccgc gcacccacac ccaggccagg
     3601 gtgttgtccg gcaccacctg gtcctggacc gcgctgatga acagggtcac gtcgtcccgg
     3661 accacaccgg cgaagtcgtc ctccacgaag tcccgggaga acccgagccg gtcggtccag
     3721 aactcgaccg ctccggcgac gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg
     3781 gccatgtata cttgtttgat taatgtggct gatagctaag ctatcgcaaa tatttgtaca
     3841 agacatgagg gggcaaaaaa ataagccaag tgtatttgtt aggcttccgt gcctgttaga
     3901 gaaatatgta aggaaacgcg tgcgctacac agcttgagca cccatcgaat tgggacgaga
     3961 acatgttaaa ctcagtactc acccgataac ccctattttc aatgcacgcc cacccagaga
     4021 cacggaatag cataaaacaa ccccatcact gccacaaaat ggacattttg cacaaaacgc
     4081 actattgact agtattcgac tatactaata agagatacct atcagacacg atatactgtg
     4141 aaaaggacat taagctcact atgcatctga aaacgctttc acctcacaac ctacttacac
     4201 acacctttcc gcttaacccg ctagctaata cgactcacta tagggcggtc ttcgagaaga
     4261 cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     4321 agtggcaccg agtcggtgcc tcgagggccg gcatggtccc agcctcctcg ctggcgccgg
     4381 ctgggcaaca tgcttcggca tggcgaatgg gactctagag ggcccctgca g
//
