LOCUS       sequence-176606-        2908 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Backbone for cloning LwCas13a guides under U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2908)
  AUTHORS   Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, 
            Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, 
            Voytas DF, Ting AY, Zhang F
  TITLE     RNA targeting with CRISPR-Cas13.
  JOURNAL   Nature. 2017 Oct 4. doi: 10.1038/nature24049.
  PUBMED    28976959
REFERENCE   2  (bases 1 to 2908)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2017 Oct 4. doi: 10.1038/nature24049."
COMMENT     Sequence Label: pC016 - LwCas13a guide expression backbone wit...
FEATURES             Location/Qualifiers
     source          1..2908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      138..726
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     627..646
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        789..1037
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     789..809
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     960..979
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     rep_origin      1173..1628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1260..1279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1470..1491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1645..1664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1764..1786
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1824..1842)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1910..2014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2015..2083,2084..2875)
                     /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(2233..2252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
       61 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
      121 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      181 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      241 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      301 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      361 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      421 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      481 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      541 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      601 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      661 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      721 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      781 cagctagcga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
      841 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
      901 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
      961 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1021 ggaaaggacg aaacaccgat ttagactacc ccaaaaacga aggggactaa aacgggtctt
     1081 cgagaagacc ttttttttga attctgatgc ggtattttct ccttacgcat ctgtgcggta
     1141 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     1201 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     1261 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1321 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1381 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1441 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1501 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     1561 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     1621 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     1681 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     1741 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     1801 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     1861 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1921 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1981 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2041 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2101 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2161 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2221 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2281 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2341 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2401 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2461 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2521 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2581 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2641 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2701 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     2761 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2821 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2881 agaccaagtt tactcatata tactttag
//
