LOCUS Exported 2812 bp ds-DNA circular SYN 25-AUG-2021 DEFINITION Cas9-mediated knockout of Ssh2 in mammalian cells. ACCESSION . VERSION . KEYWORDS Ssh2 gRNA#1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2812) AUTHORS Aihara S, Fujimoto S, Sakaguchi R, Imai T TITLE BMPR-2 gates activity-dependent stabilization of primary dendrites during mitral cell remodeling. JOURNAL Cell Rep. 2021 Jun 22;35(12):109276. doi: 10.1016/j.celrep.2021.109276. PUBMED 34161760 REFERENCE 2 (bases 1 to 2812) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 25, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..2812 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 72..312 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 72..92 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 243..262 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 341..416 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(428..452) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 658..714 /label=MCS /note="pUC18/19 multiple cloning site" primer_bind complement(727..743) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(727..743) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(740..762) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 751..767 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(775..805) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 820..841 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(1129..1717) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1209..1228) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1888..2748) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 2511..2530 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2749..41) /gene="bla" /label=AmpR promoter ORIGIN 1 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 61 ccacctgacg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 121 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata 181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 301 tgtggaaagg acgaaacacc gtagacgagc cattccctcg gttttagagc tagaaatagc 361 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 421 tttctagacc cagctttctt gtacaaagtt ggcattagat ccgctgcatt aatgaatcgg 481 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 541 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 601 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagaa 661 ttcgagctcg gtacccgggg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta 721 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 781 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 1321 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 1381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 1441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 1501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 1561 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 1621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 1681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 1741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 1801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 1861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 1921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 1981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 2041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 2101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 2161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 2221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 2281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 2341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 2401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 2461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 2521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 2581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 2641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 2701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 2761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca ta //