LOCUS Exported 3220 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION tRNA and scaffold for the assembly of GBoligomers for the first position (positon [M1_2]) of a polycistronic tRNA-gRNA regulated by the (monocot) U3 promoter (3-part multiplexing). ACCESSION . VERSION . KEYWORDS tRNA-gRNA position [M1_2] (GB1209) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3220) AUTHORS Vazquez-Vilar M, Bernabe-Orts JM, Fernandez-Del-Carmen A, Ziarsolo P, Blanca J, Granell A, Orzaez D TITLE A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. JOURNAL Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. eCollection 2016. PUBMED 26839579 REFERENCE 2 (bases 1 to 3220) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3220 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA complement(9..84) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(279..297) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(280..297) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(315..331) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(315..331) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(328..350) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 339..355 /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(363..393) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 408..429 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(546..563) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(717..1305) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(797..816) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1476..2336) /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 2099..2118 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2337..2441) /gene="bla" /label=AmpR promoter rep_origin complement(2519..2974) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2656..2677) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2868..2887 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3100..3122 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3114..3131 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3115..3131 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3138..3157 /label=T7 /note="T7 promoter, forward primer" promoter 3138..3156 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 3187..3196 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" regulatory 3191..3200 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 ctcgctcagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta 61 acttgctatt tctagctcta aaactgagac ggcgcgcgcc gtctctgcac cagccgggaa 121 tcgaacccgg gtctgtaccg tggcagggta ctattctacc actagaccac tggtgctttg 181 tttgcccgag cgagacggga tcactagtga attcgcggcc gcctgcaggt cgaccatatg 241 ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc 301 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 361 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 421 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 481 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 541 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 601 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 661 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 721 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 781 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 841 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 901 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 961 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1021 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1081 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1141 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1201 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1261 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1321 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1381 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1441 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1501 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1561 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1621 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1681 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1741 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1801 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1861 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1921 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1981 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2041 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 2101 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 2161 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 2221 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 2281 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 2341 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 2401 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 2461 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa 2521 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt 2581 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata 2641 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac 2701 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa 2761 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc 2821 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg 2881 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca 2941 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact 3001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat 3061 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa 3121 cgacggccag tgaattgtaa tacgactcac tatagggcga attgggcccg acgtcgcatg 3181 ctcccggccg ccatggcggc cgcgggaatt cgattcccgt //