LOCUS       sequence-101403-        3165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MuLE (Multiple Lentiviral Expression) Entry vector containing a U6 
            promoter and miR30-based hairpin module for shRNA expression. 
            Compatible with MultiSite Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3165)
  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 3165)
  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-miR-30 L1-L4
FEATURES             Location/Qualifiers
     source          1..3165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        733..981
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     733..753
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     904..923
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           1036..1129
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1143..1255
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(1280..1375)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1406..1425)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1407..1425)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1430..1446)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1430..1446)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1548..1567)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1559..2368
                     /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(1626..1645)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2515..3103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3004..3023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgg caaaacgcac cacgtgacgg agcgtgaccg cgcgccgagc gcgcgccagg
      721 gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat acgatacaag
      781 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
      841 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
      901 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      961 ttgtggaaag gacgaaacac cgtgctcgct tcggcagcac atatactagt cgactaggga
     1021 taacagggta attgtttgaa tgaggcttca gtactttaca gaatcgttgc ctgcacatct
     1081 tggaaacact tgctgggatt acttcttcag gttaacccaa cagaaggctc gagcaaccag
     1141 aattcaaggg gctactttag gagcaattat cttgtttact aaaactgaat accttgctat
     1201 ctctttgata catttttaca aagctgaatt aaaatggtat aaattaaatc acttttttca
     1261 attggaagac taatgcaccc aacttttcta tacaaagttg gcattataag aaagcattgc
     1321 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
     1381 ccagctgcag ggcggccgcg atatccccta tagtgagtcg tattacatgg tcatagctgt
     1441 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     1501 tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     1561 gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt
     1621 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt
     1681 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     1741 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     1801 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     1861 aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     1921 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     1981 cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     2041 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     2101 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     2161 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     2221 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     2281 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     2341 tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca
     2401 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag
     2461 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2521 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2581 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2641 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2701 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2761 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2821 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2881 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2941 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3001 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3061 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3121 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtt
//
