LOCUS Exported 3637 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-DT3T4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3637) 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 3637) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3637 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 1..76 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" gap 667..685 /estimated_length=19 primer_bind complement(733..752) /label=T7 /note="T7 promoter, forward primer" promoter complement(734..752) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(759..776) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(759..775) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(768..790) /label=M13/pUC Forward /note="In lacZ gene" CDS 913..1215 /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 1094..1113 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1564..2223 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(1630..1649) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" rep_origin 2448..3036 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2937..2956 /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 3201..3287 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 3379..3406 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" primer_bind 3414..3437 /label=pENTR-F /note="pENTR vectors, forward primer" promoter 3448..3495 /label=EM7 promoter /note="synthetic bacterial promoter " primer_bind 3515..3537 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 3534..3550 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 3534..3550 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 3568..3586 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3568..3585 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 61 ggcaccgagt cggtgctttt ttttggcaaa aattttcaga ttttttcttc atctgtagat 121 ttctgggttt ttttttccgt ttcgtgaatc ataagtgaag ttttggatgc aaatctgcgc 181 gaaaaaagtt ggacctgcaa tgagcttatt tagatagcta agacaaagtg attggtccgt 241 tcgacttgcc ttccgcacaa tacatcattt cttcttagct ttttttcttc ttcttcgttc 301 atacagtttt tttttgttta tcagcttaca ttttcttgaa ccgtagcttt cgttttcttc 361 tttttaactt tccattcgga gtttttgtat cttgtttcat agtttgtccc aggattagaa 421 tgattaggca tcgaaccttc aagaatttga ttgaataaaa catcttcatt cttaagatat 481 gaagataatc ttcaaaaggc ccctgggaat ctgaaagaag agaagcaggc ccatttatat 541 gggaaagaac aatagtattt cttatatagg cccatttaag ttgaaaacaa tcttcaaaag 601 tcccacatcg cttagataag aaaacgaagc tgagtttata tacagctaga gtcgaagtag 661 tgattgnnnn nnnnnnnnnn nnnnngttta gagaccaata atcctttggc ctatttggcc 721 tggcccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt 781 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 841 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 901 ctatacgtac ggcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt 961 gtggatgtac agagtgatat tattgacacg ccggggcgac ggatggtgat ccccctggcc 1021 agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg 1081 gatgaaagct ggcgcatgat gaccaccgat atggcaagtg tgccagtatc cgttatcggg 1141 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg 1201 ttctggggaa tataaatgtc aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt 1261 ttcacgtaga aagccagtcc gcagaaacgg accatgagtg gacataagcc tcgttcggtt 1321 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa 1381 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg 1441 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg 1501 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac 1561 atcatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa 1621 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg 1681 gatattacgg cctttttaaa gaccgtaaag aaaagtaagc acaagtttta tccggccttt 1741 attcacattc ttgcccgcct gatgaatgct catccggagt tccgtatggc aatgaaagac 1801 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact 1861 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata 1921 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt 1981 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac 2041 gtggccaata tggacaactt cttcgccccc gttttcacaa tgggcaaata ttatacgcaa 2101 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc 2161 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg 2221 taattttttt aaggcagtta ttggtgcgat cctctagcta gaaattcgtt caagccgacg 2281 ccgcttcgcg gcgcggctta actcaagcgt tagatgcact aagcacataa ttgctcacag 2341 ccaaactatc aggtcaagtc ccgcggagtt gttcggtaaa ttgtcacaac gccgcccgcc 2401 ccgttccact gagcgtcaga cccggtagaa aagatcaaag gatcttcttg agatcctttt 2461 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 2521 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 2581 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 2641 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 2701 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 2761 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 2821 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 2881 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 2941 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 3001 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 3061 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 3121 tctgtggata accgtattac cgctaggatg gatctcgggc acgtctaact actaagcgag 3181 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcggaagac tgggcctttc 3241 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg 3301 atttgaacgt tgtgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg 3361 ccaggcatca aactaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa 3421 ctcttcctgt tagttagtta cttaagtgtt gacaattaat catcggcata gtatatcggc 3481 atagtataat acgacaaggt gaggaactaa accgagcgga taacaatttc acacaggaaa 3541 cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac tcaagctatg 3601 caaggccctt aaggccaagg aataatggtc tctattg //