LOCUS       sequence-247622-        2903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hU6-driven expression of guide RNAs compatible with CasRx. 5' 
            processed DR followed by BbsI sites for guide cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2903)
  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 2903)
  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: pXR003: CasRx gRNA cloning backbone
FEATURES             Location/Qualifiers
     source          1..2903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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..286
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     rep_origin      386..841
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(473..492)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     683..704
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(858..877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     977..999
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1037..1055)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1123..1227
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1228..1296,1297..2088)
                     /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(1446..1465)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2259..2847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2748..2767
                     /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 acaccgaacc cctaccaact ggtcggggtt tgaaacgggt cttcgagaag
      301 accttttttt tgaattctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
      361 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
      421 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
      481 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      541 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      601 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      661 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
      721 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
      781 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
      841 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
      901 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
      961 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     1021 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     1081 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1141 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1201 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1261 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1321 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1381 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1441 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1501 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1561 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1621 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1681 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1741 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     1801 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1861 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1921 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     1981 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2041 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2101 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2161 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2221 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2281 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2341 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2401 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2461 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2521 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2581 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2641 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2701 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2761 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2821 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2881 gccttttgct ggccttttgc tca
//
