LOCUS Exported 3140 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Multisite gateway vector for 3' expression of a guide RNA from the U6 promoter (cassette in the reverse orientation). Contains BseRI sites for insertion of gRNA sequence. ACCESSION . VERSION . KEYWORDS p3E-rev_U6_MCS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3140) TITLE Parton Lab Plasmids for in vivo cell biology in the zebrafish REFERENCE 2 (bases 1 to 3140) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3140 /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 complement(624..748) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(755..771) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_RNA complement(784..859) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(1249..1344) /gene="mutant version of attL" /label=attL3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1381..1400) /label=T7 /note="T7 promoter, forward primer" promoter complement(1382..1400) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1405..1421) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1405..1421) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1523..1542) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1534..2343 /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(1601..1620) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 2490..3078 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2979..2998 /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac 601 taataccatc taagtagttg attcatagtg actgcatatg ttgtgtttta cagtattatg 661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 721 ctcgttcaac tttcttgtac aaagtggata attcactggc cgtcgtttta cggtaccaaa 781 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg 841 ctatttctag ctctaaaacg ctactgactc ctcgtcactg aggagtgcta gccgaaccaa 901 gagctggagg gagagctata tataccaggg acttctgggt atgtttttgg gaggtggtga 961 gtgactaaac cacttattca gctcccttag atcaagtctg accctatatc atggtgacat 1021 aaacctgcaa actgataaaa cctgaaagat ctcaaatcca gagtttgtgt gagggtttac 1081 cgtggtattt ttcaaacttt aaagcatatg caaaattatc tggtctcggc ttgagtgatt 1141 gggtgtctcg gtgtgatgca gggacgtttt cagtgacgtg tccttctccc tcccccacag 1201 gcatgcgcag aacattcccc cctccttgat gaccagaaca aaagacgcca actttattat 1261 acaaagttgg cattataaaa aagcattgct tatcaatttg ttgcaacgaa caggtcacta 1321 tcagtcaaaa taaaatcatt atttggagct ccatggtagc gttaacgcgg ccgcgatatc 1381 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct 1441 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg 1501 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg 1561 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat 1621 aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag 1681 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga 1741 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct 1801 gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa 1861 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg 1921 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag 1981 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat 2041 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat 2101 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta 2161 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc 2221 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat 2281 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc 2341 taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac 2401 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca 2461 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2521 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2581 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 2641 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 2701 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2761 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2821 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2881 cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 2941 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 3001 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 3061 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 3121 tggccttttg ctcacatgtt //