LOCUS       sequence-199180-        2962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning guides that are compatible with LwaCas13a. 
            Contains a 5' direct repeat. Clone using BbsI (BpiI). F overhang 
            AAAC. R overhang AAAA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2962)
  AUTHORS   Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung 
            J, Zhang F
  TITLE     RNA editing with CRISPR-Cas13.
  JOURNAL   Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
  PUBMED    29070703
REFERENCE   2  (bases 1 to 2962)
  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: "Science. 
            2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180."
COMMENT     Sequence Label: pC0040-LwaCas13a crRNA backbone
FEATURES             Location/Qualifiers
     source          1..2962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(38..626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(118..137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(797..1588,1589..1657))
                     /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     1420..1439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1658..1762)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1830..1848
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1886..1908)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2008..2027
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2221..2243
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2235..2252
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2236..2252
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(2336..2584)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2394..2413)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2564..2584)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2598..2614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2598..2614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2611..2633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2622..2638
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2646..2652,2653..2670,2671..2676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2691..2712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2829..2846)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
       61 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      121 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
      181 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
      241 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
      301 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
      361 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      421 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
      481 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
      541 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
      601 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
      661 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
      721 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
      781 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
      841 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
      901 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
      961 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1021 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1081 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     1141 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1201 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     1261 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     1321 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     1381 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     1441 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     1501 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     1561 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     1621 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     1681 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     1741 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     1801 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     1861 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     1921 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     1981 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2041 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     2101 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     2161 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     2221 cccagtcacg acgttgtaaa acgacggcca gtgaattcga gctcggtacc aaaaaattgt
     2281 cttcgtccca ggaagacatg ttttagtccc cttcgttttt ggggtagtct aaatcggtgt
     2341 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     2401 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     2461 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa
     2521 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     2581 cctctggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2641 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2701 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2761 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2821 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2881 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2941 atgtgagcaa aaggccagca aa
//
