LOCUS       sequence-112709-        3783 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 3783)
  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 3783)
  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 R4-R3
FEATURES             Location/Qualifiers
     source          1..3783
                     /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=attR3
                     /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    complement(2650..2773)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2817..3065
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2817..2837
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2988..3007
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3092..3751)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3666..3685
                     /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 aataaagttg 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 ggcccctaca ggtcactaat accatctaag
     2641 tagttgattc atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta
     2701 tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg ttcaactttt
     2761 ctatacaaag ttgctatcga tcacgagact agcctcgagc ggccgccccc ttcaccgagg
     2821 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2881 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2941 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     3001 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     3061 acaccggtgt gcaggtgagt gatccagatc tttacgcccc gccctgccac tcatcgcagt
     3121 actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa
     3181 cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga
     3241 aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca
     3301 cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca
     3361 ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt
     3421 cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac
     3481 aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaattccg
     3541 gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat
     3601 ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac
     3661 attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa
     3721 cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg
     3781 acg
//
