LOCUS       sequence-101404-        3162 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 3162)
  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 3162)
  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 L5-L2
FEATURES             Location/Qualifiers
     source          1..3162
                     /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    570..665
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        730..978
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     730..750
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     901..920
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           1033..1126
                     /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           1140..1252
                     /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(1274..1373)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1403..1422)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1404..1422)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1427..1443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1427..1443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1545..1564)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1556..2365
                     /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(1623..1642)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2512..3100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3001..3020
                     /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 tgtatacaaa
      661 agttgggcaa aacgcaccac gtgacggagc gtgaccgcgc gccgagcgcg cgccagggtc
      721 gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      781 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
      841 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
      901 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
      961 tggaaaggac gaaacaccgt gctcgcttcg gcagcacata tactagtcga ctagggataa
     1021 cagggtaatt gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg
     1081 aaacacttgc tgggattact tcttcaggtt aacccaacag aaggctcgag caaccagaat
     1141 tcaaggggct actttaggag caattatctt gtttactaaa actgaatacc ttgctatctc
     1201 tttgatacat ttttacaaag ctgaattaaa atggtataaa ttaaatcact tttttcaatt
     1261 ggaagactaa tgtacccagc tttcttgtac aaagttggca ttataagaaa gcattgctta
     1321 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca
     1381 gctgcagggc ggccgcgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
     1441 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     1501 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     1561 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     1621 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     1681 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     1741 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     1801 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     1861 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     1921 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     1981 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     2041 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     2101 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     2161 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     2221 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     2281 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     2341 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     2401 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     2461 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2521 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2581 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2641 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2701 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2761 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2821 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2881 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2941 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3001 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3061 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3121 tttacggttc ctggcctttt gctggccttt tgctcacatg tt
//
