LOCUS       40924_7811        2962 bp DNA     circular SYN 20-DEC-2021
DEFINITION  For cloning of guide RNAs compatible with PspCas13b. Contains a 3' 
            direct repeat. Clone using BbsI (BpiI). F overhang cacc. R overhang 
            caac.
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)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2962)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     128..147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     381..398
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    515..536
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        551..581
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    589..605
                     /label=lac operator
                     /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)."
     primer_bind     613..629
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        643..883
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(975..991)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1200..1219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     1319..1341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1379..1397)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        1465..1569
                     /label=AmpR promoter
     CDS             1570..2427
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2601..2962
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
       61 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      121 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      181 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      241 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      301 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      361 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
      421 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      481 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      541 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      601 aacaatttca cgcaggaaac agctatgacc atgattacgc cagagggcct atttcccatg
      661 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      721 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      781 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      841 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac catgtcttcc
      901 tgggacgaag acaagttgtg gaaggtccag ttttgagggg ctattacaac ttttttggta
      961 ccgagctcga attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     1021 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     1081 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     1141 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     1201 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     1261 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     1321 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     1381 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1441 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1501 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1561 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1621 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1681 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1741 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1801 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1861 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1921 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1981 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2041 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2101 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2161 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2221 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2281 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2341 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2401 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2461 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2521 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2581 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2641 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2701 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2761 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2821 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2881 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2941 cccagcttgg agcgaacgac ct
//
