LOCUS Exported 3192 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PCR template to construct polycistronic tRNA-gRNA. ACCESSION . VERSION . KEYWORDS pGTR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3192) AUTHORS Xie K, Minkenberg B, Yang Y TITLE Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. JOURNAL Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PUBMED 25733849 REFERENCE 2 (bases 1 to 3192) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3192 /organism="synthetic DNA construct" /mol_type="other DNA" regulatory 33..42 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 37..46 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_RNA complement(145..220) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(317..335) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(318..335) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(353..369) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(353..369) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(366..388) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 377..393 /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(401..431) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 446..467 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(584..601) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(755..1343) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(835..854) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1514..2374) /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 2137..2156 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2375..2479) /gene="bla" /label=AmpR promoter rep_origin complement(2557..3012) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2694..2715) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2906..2925 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3138..3160 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3152..3169 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3153..3169 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3176..3 /label=T7 /note="T7 promoter, forward primer" promoter 3176..2 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 tctgccatgc accagccggg aatcgaaccc gggtctgtac cgtggcaggg tactattcta 121 ccactagacc actggtgctt tgttgcaccg actcggtgcc actttttcaa gttgataacg 181 gactagcctt attttaactt gctatttcta gctctaaaac cgagacctcg gtctccaatc 241 actagtgaat tcgcggccgc ctgcaggtcg accatatggg agagctccca acgcgttgga 301 tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca tggtcatagc 361 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 421 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 481 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 541 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 601 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 661 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 721 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 781 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 841 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 901 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 961 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1021 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1081 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1141 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 1201 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 1261 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1321 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1381 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1441 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1501 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1561 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1621 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1681 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1741 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1801 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1861 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1921 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1981 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 2041 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 2101 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 2161 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 2221 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 2281 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2341 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2401 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2461 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata 2521 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt 2581 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg 2641 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt 2701 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct 2761 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt 2821 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa 2881 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc 2941 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc 3001 tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 3061 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 3121 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata 3181 cgactcacta ta //