LOCUS       sequence-112700-        3725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MuLE (Multiple Lentiviral Expression) Entry vector containing a U6 
            promoter and sgRNA scaffold module for gRNA expression. Compatible 
            with MultiSite Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3725)
  AUTHORS   Albers J, Danzer C, Rechsteiner M, Lehmann H, Brandt LP, Hejhal T, 
            Catalano A, Busenhart P, Goncalves AF, Brandt S, Bode PK, 
            Bode-Lesniewska B, Wild PJ, Frew IJ
  TITLE     A versatile modular vector system for rapid combinatorial mammalian 
            genetics.
  JOURNAL   J Clin Invest. 2015 Mar 9. pii: 79743. doi: 10.1172/JCI79743.
  PUBMED    25751063
REFERENCE   2  (bases 1 to 3725)
  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: "J Clin 
            Invest. 2015 Mar 9. pii: 79743. doi: 10.1172/JCI79743."
COMMENT     Sequence Label: pMuLE ENTR U6 stuffer sgRNA scaffold L1-R5
FEATURES             Location/Qualifiers
     source          1..3725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        19..94
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    110..233
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(288..307)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(289..307)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(312..328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(312..328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(430..449)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             441..1250
                     /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(508..527)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1397..1985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1886..1905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2139..2156
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2315..2342)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2434..2520)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2569..2591
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2583..2600
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2584..2600
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2616..2715
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2759..3007
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2759..2779
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2930..2949
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3034..3693)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3608..3627
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 gatcaagaac ctgctgacgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
       61 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattcgac aactttgtat
      121 acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg
      181 cataaaaaac agactacata atactgtaaa acacaacata tccagtcact atgaatcaac
      241 tacttagatg gtattagtga cctgtactgc agggcggccg cgatatcccc tatagtgagt
      301 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
      361 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
      421 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
      481 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
      541 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
      601 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
      661 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
      721 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
      781 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
      841 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
      901 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
      961 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     1021 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     1081 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     1141 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     1201 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     1261 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat
     1321 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     1381 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1441 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1501 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1561 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1621 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1681 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1741 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1801 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1861 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1921 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1981 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2041 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2101 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2161 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2221 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
     2281 gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     2341 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     2401 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     2461 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
     2521 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac
     2581 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga
     2641 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt
     2701 acaaaaaagc aggctctatc gatcacgaga ctagcctcga gcggccgccc ccttcaccga
     2761 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     2821 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     2881 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     2941 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     3001 aaacaccggt gtgcaggtga gtgatccaga tctttacgcc ccgccctgcc actcatcgca
     3061 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
     3121 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt
     3181 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
     3241 cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc
     3301 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
     3361 gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
     3421 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaattc
     3481 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
     3541 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt
     3601 acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc
     3661 aacggtggta tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct
     3721 cgacg
//
