LOCUS Exported 2476 bp ds-DNA circular SYN 03-AUG-2021 DEFINITION single guide RNA (sgRNA) and Prime editing guide RNA (pegRNA) cloning and mammalian cell expression. BbsI cloning for sgRNAs and BbsI/PstI for pegRNAs.. ACCESSION . VERSION . KEYWORDS pHSG1C3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2476) AUTHORS Standage-Beier K, Tekel SJ, Brafman DA, Wang X TITLE Prime Editing Guide RNA Design Automation Using PINE-CONE. JOURNAL ACS Synth Biol. 2021 Feb 19;10(2):422-427. doi: 10.1021/acssynbio.0c00445. Epub 2021 Jan 19. PUBMED 33464043 REFERENCE 2 (bases 1 to 2476) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 3, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..2476 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 7..247 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7..27 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 178..197 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 274..349 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 450..507 /label=his operon terminator /note="This putative transcriptin terminator from the E. coli his operon has a 2-bp deletion introduced during synthesis. Its efficiency has not been determined." primer_bind complement(531..548) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(702..1290) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(782..801) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 1473..1567 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(1588..2247) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2162..2181 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" primer_bind 2409..2427 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 2431..2474 /label=bacterial terminator /note="putative bacterial transcription terminator" ORIGIN 1 gaattcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 241 aaaggacgaa acaccgggtc ttcgagaaga cctgttttag agctagaaat agcaagttaa 301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt 361 agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct 421 gcagacaaat ctagtagcgg ccgctgcagt ccggcaaaaa agggcaaggt gtcaccaccc 481 tgcccttttt ctttaaaacc gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact 541 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 601 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 661 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccacag gctccgcccc 721 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 781 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 841 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 901 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 961 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 1021 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 1081 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 1141 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 1201 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 1261 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 1321 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 1381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 1441 gagtaaactt ggtctgacag ctcgaggctt ggattctcac caataaaaaa cgcccggcgg 1501 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac 1561 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg 1621 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg 1681 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac 1741 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca 1801 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt 1861 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg 1921 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg 1981 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg 2041 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt 2101 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg 2161 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt 2221 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa 2281 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac 2341 gtgcccgatc aactcgagtg ccacctgacg tctaagaaac cattattatc atgacattaa 2401 cctataaaaa taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg 2461 ctaaggatga tttctg //