LOCUS Exported 3481 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS PU6::unc-119_sgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3481) AUTHORS Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA TITLE Heritable genome editing in C. elegans via a CRISPR-Cas9 system. JOURNAL Nat Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532. PUBMED 23817069 REFERENCE 2 (bases 1 to 3481) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3481 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 364..386 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 378..395 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 379..395 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 402..901 /label=CeU6 promoter /note="promoter for the C. elegans K09B11.12 U6 snRNA gene (Friedland et al., 2013)" misc_RNA 922..997 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 1005..1241 /label=3' CeU6 /note="region downstream of the C. elegans K09B11.12 U6 snRNA gene" primer_bind complement(1260..1276) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1260..1276) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1273..1295) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1284..1300 /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(1308..1338) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1353..1374 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1491..1508) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1662..2250) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1742..1761) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2421..3281) /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 3044..3063 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3282..3386) /gene="bla" /label=AmpR promoter primer_bind 3454..3472 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cctccaagaa ctcgtacaaa 421 aatgctctga agtaggtctc gagatcaata ctgggaagac gaaagtcttg cgaaaccgat 481 tcgctgaccc cagtaaagtc tacttcggta gcccttcccc caccacccag ctcgacgacg 541 tcgacgagta catctacctc ggtcgtcaaa tcaacgccca aaacaacttg atgccggaaa 601 tccaccgaag acgtcgagca gcctgggctg cattcaatgg aatcaagaat gccaccgact 661 ccatcaccga caagaagatt cgtgcgaatc tgttcgactc aattgtcctt ccagcgctca 721 cctacgtttc agaagcctgg acattcacca aagctctatc cgaacgagta cgaatcacac 781 atgcctcccc cacaaattat tgatctacat ccccatttgg tatagtgtct tacaggactc 841 tcaatatgtc agctgcatga taaatggtcc ctatataatt gaattgcaaa tctaaatgtt 901 tgaattttct gaaattaaag agttttagag ctagaaatag caagttaaaa taaggctagt 961 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaataa tattctagat 1021 atcccttttt gaaactaaaa tgaaatactc taaatttttg agttcaatgt ttcaactaat 1081 cttggttaaa ggtaaattcc cggagggacg attttaaagt tgccagagtt gattaaattg 1141 gtccaaaaaa ttttattagg aaactcgaaa aaaaattgat tttttttgac aatattccaa 1201 agaaaattac aaaatctcga cgccaaacat agtcggctgt gaagcttggc gtaatcatgg 1261 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 1321 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 1381 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 1441 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 1501 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 1561 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 1621 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 1681 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 1741 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 1801 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 1861 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 1921 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 1981 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 2041 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 2101 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 2161 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 2221 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 2281 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 2341 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 2401 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 2461 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 2521 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 2581 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 2641 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 2701 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 2761 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 2821 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 2881 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 2941 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 3001 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 3061 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 3121 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 3181 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 3241 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 3301 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 3361 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 3421 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 3481 c //