LOCUS Exported 4178 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pMAGIC L5-L4 entry plasmid, contains empty human U6-driven SaCas9 gRNA scaffold and EF1a promoter for 4-component MultiSite Gateway Pro assembly. Protospacer motif can be inserted after BsaI digestion. ACCESSION . VERSION . KEYWORDS KB601: pMAGIC (L5-L4) hU6::SaCas9 gRNA scaffold; EF1a promoter SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4178) AUTHORS Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, Newgard CB TITLE Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. JOURNAL Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286. PUBMED 30590691 REFERENCE 2 (bases 1 to 4178) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4178 /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 570..665 /gene="mutant version of attL" /label=attL5 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 674..914 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 674..694 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 845..864 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 1064..2242 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 1295..2233 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 2190..2210 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" promoter 2259..2277 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind complement(2293..2388) /gene="mutant version of attL" /label=attL4 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter complement(2420..2438) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2443..2459) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2443..2459) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2561..2580) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2572..3381 /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(2639..2658) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 3528..4116 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4017..4036 /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 agttgtggta cctgagggcc tatttcccat gattccttca tatttgcata tacgatacaa 721 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa 781 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa 841 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat 901 cttgtggaaa ggacgaaaca ccggagacca cggcaggtct cagtttaagt actctgtgct 961 ggaaacagca cagaatctac taaacaaggc aaaatgccgt gtttatctcg tcaacttgtt 1021 ggcgagattt ttgcggccgc tgtacatacg actctagacg tgaggctccg gtgcccgtca 1081 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg 1141 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct 1201 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt 1261 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg 1321 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca 1381 gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg 1441 cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg 1501 ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc 1561 catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa 1621 tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc 1681 ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat 1741 cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg 1801 tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag 1861 atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga 1921 gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc 1981 atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg 2041 gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga 2101 gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc 2161 cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt 2221 tcttccattt caggtgtcgt gaggaattag cttggtacta atacgactca ctatagggag 2281 acgaattcca cccaactttt ctatacaaag ttggcattat aagaaagcat tgcttatcaa 2341 tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg 2401 cagggcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg 2461 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca 2521 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat 2581 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg 2641 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg 2701 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt 2761 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag 2821 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca 2881 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca 2941 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc 3001 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat 3061 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt 3121 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt 3181 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga 3241 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa 3301 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg 3361 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt 3421 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg 3481 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 3541 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 3601 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 3661 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 3721 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 3781 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 3841 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 3901 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 3961 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4021 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4081 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4141 cggttcctgg ccttttgctg gccttttgct cacatgtt //