LOCUS       Exported                2945 bp ds-DNA     circular SYN 14-FEB-2022
DEFINITION  hU6-driven expression of guide RNAs compatible with CasRx. Contains 
            BbsI sites for guide cloning flanked by 5' and 3' full-length DRs.
ACCESSION   .
VERSION     .
KEYWORDS    pXR004: CasRx pre-gRNA cloning backbone
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2945)
  AUTHORS   Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD
  TITLE     Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR 
            Effectors.
  JOURNAL   Cell. 2018 Mar 8. pii: S0092-8674(18)30207-1. doi: 
            10.1016/j.cell.2018.02.033.
  PUBMED    29551272
REFERENCE   2  (bases 1 to 2945)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 14, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2945
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        7..247
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        257..292
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        311..346
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     rep_origin      428..883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(515..534)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     725..746
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(900..919)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1019..1041
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1079..1097)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1165..1269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1270..2130
                     /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(1488..1507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2301..2889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2790..2809
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctagcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgcaag taaaccccta ccaactggtc ggggtttgaa acgggtcttc
      301 gagaagacct caagtaaacc cctaccaact ggtcggggtt tgaaactttt tttgaattct
      361 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
      421 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      481 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
      541 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      601 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
      661 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      721 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
      781 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
      841 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
      901 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
      961 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     1021 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     1081 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1141 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1201 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1261 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1321 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1381 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1441 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1501 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1561 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1621 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1681 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1741 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1801 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1861 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1921 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     1981 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2041 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2101 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2161 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2221 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2281 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2341 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2401 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2461 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2521 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2581 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2641 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2701 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2761 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2821 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2881 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2941 gctca
//
