LOCUS Exported 3544 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR/Cas based plant genome editing and gene regulation; used as template for making expression cassette with multiple gRNA target sites. ACCESSION . VERSION . KEYWORDS pCBC-DT1T2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3544) AUTHORS Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ TITLE A CRISPR/Cas9 toolkit for multiplex genome editing in plants. JOURNAL BMC Plant Biol. 2014 Nov 29;14(1):327. PUBMED 25432517 REFERENCE 2 (bases 1 to 3544) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3544 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 20..95 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(644..662) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(645..662) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(680..696) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(680..696) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(693..715) /label=M13/pUC Reverse /note="In lacZ gene" promoter complement(735..782) /label=EM7 promoter /note="synthetic bacterial promoter " primer_bind complement(793..816) /label=pENTR-F /note="pENTR vectors, forward primer" terminator 824..851 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 943..1029 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" rep_origin complement(1194..1782) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1274..1293) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2007..2666) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2581..2600 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS complement(3015..3317) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(3117..3136) /label=ccdB-fwd /note="ccdB gene, forward primer" primer_bind 3440..3462 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 3454..3471 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 3455..3471 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 3478..3497 /label=T7 /note="T7 promoter, forward primer" promoter 3478..3496 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gacggccatg gcggttctcg ttttagagct agaaatagca agttaaaata aggctagtcc 61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tttgcaaaat tttccagatc 121 gatttcttct tcctctgttc ttcggcgttc aatttctggg gttttctctt cgttttctgt 181 aactgaaacc taaaatttga cctaaaaaaa atctcaaata atatgattca gtggttttgt 241 acttttcagt tagttgagtt ttgcagttcc gatgagataa accaatatta atccaaacta 301 ctgcagcctg acagacaaat gaggatgcaa acaattttaa agtttatcta acgctagctg 361 ttttgtttct tctctctggt gcaccaacga cggcgttttc tcaatcataa agaggcttgt 421 tttacttaag gccaataatg ttgatggatc gaaagaagag ggcttttaat aaacgagccc 481 gtttaagctg taaacgatgt caaaaacatc ccacatcgtt cagttgaaaa tagaagctct 541 gtttatatat tggtagagtc gactaagaga ttggcaaagt tatacaatgg cagtttagag 601 accaataatc cttggcctta agggccttgc atagcttgag tattctatag tgtcacctaa 661 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcggtt 721 tagttcctca ccttgtcgta ttatactatg ccgatatact atgccgatga ttaattgtca 781 acacttaagt aactaactaa caggaagagt ttgtagaaac gcaaaaaggc catccgtcag 841 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc 901 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag 961 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg 1021 ttttatttga tgcctggcag ttccctactc tcgcttagta gttagacgtg cccgagatcc 1081 atcctagcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 1141 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 1201 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 1261 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 1321 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1381 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 1441 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 1501 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 1561 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 1621 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 1681 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 1741 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 1801 tctacggggt ctgacgctca gtggaacggg gcgggcggcg ttgtgacaat ttaccgaaca 1861 actccgcggg acttgacctg atagtttggc tgtgagcaat tatgtgctta gtgcatctaa 1921 cgcttgagtt aagccgcgcc gcgaagcggc gtcggcttga acgaatttct agctagagga 1981 tcgcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt 2041 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga 2101 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat tgtgaaaacg 2161 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag 2221 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt 2281 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg 2341 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg 2401 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ctccggatga 2461 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttacttttc 2521 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga 2581 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg 2641 gtatatccag tgattttttt ctccatgatg tttaactttg ttttagggcg actgccctgc 2701 tgcgtaacat cgttgctgct ccataacatc aaacatcgac ccacggcgta acgcgcttgc 2761 tgcttggatg cccgaggcat agactgtacc ccaaaaaaac agtcataaca agccatgaaa 2821 accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag 2881 cgcatacgct acttgcatta cagcttacga accgaacgag gcttatgtcc actcatggtc 2941 cgtttctgcg gactggcttt ctacgtgaaa aggatctagg tgaagatcct ttttgataat 3001 ctcatgcctg acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt 3061 tcgcggtggc tgagatcagc cacttcttcc ccgataacgg atactggcac acttgccata 3121 tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg 3181 gagactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgccccggc 3241 gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag 3301 gtgtaaacct taaactgccg tacgtatagg ctgcgcaact gttgggaagg gcgatcggtg 3361 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 3421 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa 3481 tacgactcac tatagggcga attgggccag gccaaatagg ccaaaggaat aatggtctct 3541 attg //