LOCUS       sequence-354498-        2945 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2018 
            Mar 8. pii: S0092-8674(18)30207-1. doi: 10.1016/j.cell.2018.02.033."
COMMENT     Sequence Label: pXR004: CasRx pre-gRNA cloning backbone
FEATURES             Location/Qualifiers
     source          1..2945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        16..51
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     rep_origin      133..588
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(220..239)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     430..451
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(605..624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     724..746
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(784..802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        870..974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(975..1043,1044..1835)
                     /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(1193..1212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2006..2594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2495..2514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2657..2905
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2657..2677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2828..2847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2907..2942
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
ORIGIN
        1 tcttcgagaa gacctcaagt aaacccctac caactggtcg gggtttgaaa ctttttttga
       61 attctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
      121 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      181 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
      241 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      301 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
      361 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      421 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
      481 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
      541 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
      601 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      661 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
      721 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
      781 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      841 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      901 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      961 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1021 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1081 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1141 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1201 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1261 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1321 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1381 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1441 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1501 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1561 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1621 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1681 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1741 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1801 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1861 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1921 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1981 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2041 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2101 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2161 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2221 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2281 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2341 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2401 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2461 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2521 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2581 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2641 cttttgctca gctagcgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2701 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2761 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2821 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2881 tatcttgtgg aaaggacgaa acaccgcaag taaaccccta ccaactggtc ggggtttgaa
     2941 acggg
//
