LOCUS       sequence-224177-        3140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway vector for 3' expression of a guide RNA from the 
            U6 promoter (cassette in the reverse orientation). Contains BseRI 
            sites for insertion of gRNA sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3140)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 3140)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: p3E-rev_U6_MCS
FEATURES             Location/Qualifiers
     source          1..3140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1..124)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(132..148)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(161..236)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(626..721)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(758..777)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(759..777)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(782..798)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(782..798)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(900..919)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             911..1720
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(978..997)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1867..2455
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2356..2375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2609..2626
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2785..2812)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2904..2990)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3039..3061
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3054..3070
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 catagtgact gcatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc
       61 taatttaata tattgatatt tatatcattt tacgtttctc gttcaacttt cttgtacaaa
      121 gtggataatt cactggccgt cgttttacgg taccaaaaaa gcaccgactc ggtgccactt
      181 tttcaagttg ataacggact agccttattt taacttgcta tttctagctc taaaacgcta
      241 ctgactcctc gtcactgagg agtgctagcc gaaccaagag ctggagggag agctatatat
      301 accagggact tctgggtatg tttttgggag gtggtgagtg actaaaccac ttattcagct
      361 cccttagatc aagtctgacc ctatatcatg gtgacataaa cctgcaaact gataaaacct
      421 gaaagatctc aaatccagag tttgtgtgag ggtttaccgt ggtatttttc aaactttaaa
      481 gcatatgcaa aattatctgg tctcggcttg agtgattggg tgtctcggtg tgatgcaggg
      541 acgttttcag tgacgtgtcc ttctccctcc cccacaggca tgcgcagaac attcccccct
      601 ccttgatgac cagaacaaaa gacgccaact ttattataca aagttggcat tataaaaaag
      661 cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
      721 tggagctcca tggtagcgtt aacgcggccg cgatatcccc tatagtgagt cgtattacat
      781 ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc
      841 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
      901 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
      961 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     1021 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     1081 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     1141 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     1201 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     1261 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     1321 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     1381 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     1441 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     1501 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     1561 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     1621 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     1681 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     1741 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     1801 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2221 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2281 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2341 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2401 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2461 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2521 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2581 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     2641 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2701 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgctag
     2761 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
     2821 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
     2881 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     2941 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
     3001 gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac gttgtaaaac
     3061 gacggccagt cttaagctcg ggccctgcag ctctagagct cgaattctac aggtcactaa
     3121 taccatctaa gtagttggtt
//
