LOCUS Exported 3214 bp ds-DNA circular SYN 13-MAY-2021 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 pMuLE ENTR U6-miR-30 L1-R5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3214) 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 3214) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3214 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 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..973 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 1277..1400 /gene="mutant version of attR" /label=attR5 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1455..1474) /label=T7 /note="T7 promoter, forward primer" promoter complement(1456..1474) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1479..1495) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1479..1495) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1597..1616) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1608..2417 /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(1675..1694) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 2564..3152 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3053..3072 /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 taatgacaac tttgtataca aaagttgaac gagaaacgta aaatgatata 1321 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca 1381 caacatatcc agtcactatg aatcaactac ttagatggta ttagtgacct gtactgcagg 1441 gcggccgcga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc 1501 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat 1561 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc 1621 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata 1681 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc 1741 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag 1801 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt 1861 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat 1921 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt 1981 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat 2041 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg 2101 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc 2161 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg 2221 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc 2281 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc 2341 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc 2401 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc 2461 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt 2521 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2581 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2641 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2701 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2761 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2821 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2881 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 2941 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 3001 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3061 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3121 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3181 tcctggcctt ttgctggcct tttgctcaca tgtt //