LOCUS Exported 3015 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Gateway entry vector containing attL3 and attL2 sites, U6 promoter, BsaI sites for protospacer ligation, and sgRNA.. ACCESSION . VERSION . KEYWORDS pENTR-PpU6P-sgRNA-L3L2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3015) AUTHORS Mallett DR, Chang M, Cheng X, Bezanilla M TITLE Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens. JOURNAL Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. eCollection 2019 Sep. PUBMED 31523744 REFERENCE 2 (bases 1 to 3015) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3015 /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=attL3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" protein_bind complement(1127..1226) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1256..1275) /label=T7 /note="T7 promoter, forward primer" promoter complement(1257..1275) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1280..1296) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1280..1296) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1398..1417) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1409..2218 /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(1476..1495) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 2365..2953 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2854..2873 /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 tgtataataa 661 agttgcccct tcaccgtcag atgctactac taaaaaaaag caccgactcg gtgccacttt 721 ttcaagttga taacggacta gccttatttt aacttgctat gcttttcagc atagctctaa 781 aactgagacc ttggtctcta tgggttgtaa gtcctccacc ttcctatata cccgccttca 841 cacccacctc cattccatgt gggttatatc cctcgctcca tcccttttct cccccccccc 901 tcgaaacccg gtcgcgtcca ctcggccagc cacccagctg gacccactca ctcactgcac 961 cctttctgct tgtgctctcc cgctgccgct ccttcgggat atccccttct ccgccaccgc 1021 aaggacgccg ccgtggaaga accacaccat cacatcacat cacatctaca cccatcgtcg 1081 ctaacctgtc gcaacacgtc tgcttcaatg gacgaattcg agctctaccc agctttcttg 1141 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact 1201 atcagtcaaa ataaaatcat tatttgccat ccagctgcag ggcggccgcg atatccccta 1261 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa 1321 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc 1381 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc 1441 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt 1501 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt 1561 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa 1621 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga 1681 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata 1741 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc 1801 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca 1861 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg 1921 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt 1981 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg 2041 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg 2101 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat 2161 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc 2221 agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc 2281 aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc 2341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 2401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 2461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 2521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 2581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 2641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 2701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 2761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 2821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 2881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 2941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 3001 ttttgctcac atgtt //