LOCUS       sequence-112703-        3676 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 3676)
  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 3676)
  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-L4
FEATURES             Location/Qualifiers
     source          1..3676
                     /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    complement(113..208)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(239..258)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(240..258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(263..279)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(263..279)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(381..400)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             392..1201
                     /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(459..478)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1348..1936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1837..1856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2090..2107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2266..2293)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2385..2471)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2520..2542
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2534..2551
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2535..2551
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2567..2666
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2710..2958
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2710..2730
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2881..2900
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2985..3644)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3559..3578
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 gatcaagaac ctgctgacgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
       61 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattcgca cccaactttt
      121 ctatacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
      181 actatcagtc aaaataaaat cattatttgc catccagctg cagggcggcc gcgatatccc
      241 ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca
      301 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
      361 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
      421 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
      481 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
      541 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
      601 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
      661 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
      721 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
      781 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
      841 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
      901 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
      961 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     1021 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     1081 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     1141 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     1201 atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg
     1261 cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga
     1321 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1381 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1441 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1501 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1561 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1621 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1681 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1741 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1801 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1861 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1921 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1981 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     2041 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     2101 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2161 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2221 aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc
     2281 aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac
     2341 cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg
     2401 agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt
     2461 cgttttattt gatgcctggc agttccctac tctcgcgtta acgctagcat ggatgttttc
     2521 ccagtcacga cgttgtaaaa cgacggccag tcttaagctc gggccccaaa taatgatttt
     2581 attttgactg atagtgacct gttcgttgca acaaattgat gagcaatgct tttttataat
     2641 gccaactttg tacaaaaaag caggctctat cgatcacgag actagcctcg agcggccgcc
     2701 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2761 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2821 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2881 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2941 tggaaaggac gaaacaccgg tgtgcaggtg agtgatccag atctttacgc cccgccctgc
     3001 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
     3061 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     3121 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     3181 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     3241 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     3301 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     3361 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     3421 atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     3481 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     3541 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     3601 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     3661 cctgaaaatc tcgacg
//
