LOCUS Exported 4781 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid encoding the Triplex/gRNA architecture.This is a modified form of the original plasmid described in the paper (Construct 3). mKate2 was replaced with dsRed2 because of distribution issues.. ACCESSION . VERSION . KEYWORDS CMVp-dsRed2-Triplex-28-gRNA1-28-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4781) AUTHORS Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK TITLE Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells. JOURNAL Mol Cell. 2014 May 14. pii: S1097-2765(14)00355-4. doi: 10.1016/j.molcel.2014.04.022. PUBMED 24837679 REFERENCE 2 (bases 1 to 4781) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4781 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(108..125) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(279..867) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(359..378) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1038..1898) /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 1661..1680 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1899..2003) /gene="bla" /label=AmpR promoter rep_origin 2030..2485 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2117..2136) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2327..2348 /label=F1ori-F /note="F1 origin, forward primer" enhancer 2674..2977 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2978..3181 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 3132..3152 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 3210..3219 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3216..3893 /codon_start=1 /product="improved tetrameric variant of DsRed fluorescent protein" /label=DsRed2 /note="mammalian codon-optimized" /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH LFL" primer_bind complement(3396..3416) /label=DsRed1-N /note="DsRed1, reverse primer" primer_bind 3808..3831 /label=DsRed1-C /note="DsRed1, forward primer" protein_bind 4068..4082 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" misc_RNA 4111..4186 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind 4200..4214 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" primer_bind complement(4477..4496) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4521..4568 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" ORIGIN 1 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct 61 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac 121 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 181 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 241 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 301 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 361 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 421 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 481 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 541 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 601 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 661 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 721 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 781 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 841 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 901 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 961 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 1021 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 1081 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 1141 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 1201 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 1261 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 1321 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 1381 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 1441 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 1501 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 1561 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 1621 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 1681 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 1741 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 1801 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 1861 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 1921 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 1981 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt 2041 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 2101 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 2161 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 2221 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 2281 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 2341 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 2401 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 2461 aacaaaatat taacgtttac aatttcccat tcgccattca ggctgcgcaa ctgttgggaa 2521 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt 2581 aatattaagg tacgggaggt acttggaggt acctagttat taatagtaat caattacggg 2641 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 2701 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 2761 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 2821 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 2881 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 2941 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 3001 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 3061 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 3121 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 3181 tggtttagtg aaccgtcaga tccgagctcg ccaccatggc ctcctccgag aacgtcatca 3241 ccgagttcat gcgcttcaag gtgcgcatgg agggcaccgt gaacggccac gagttcgaga 3301 tcgagggcga gggcgagggc cgcccctacg agggccacaa caccgtgaag ctgaaggtga 3361 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct 3421 ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg tccttccccg 3481 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggcg accgtgaccc 3541 aggactcctc cctgcaggac ggctgcttca tctacaaggt gaagttcatc ggcgtgaact 3601 tcccctccga cggccccgtg atgcagaaga agaccatggg ctgggaggcc tccaccgagc 3661 gcctgtaccc ccgcgacggc gtgctgaagg gcgagaccca caaggccctg aagctgaagg 3721 acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag cccgtgcagc 3781 tgcccggcta ctactacgtg gacgccaagc tggacatcac ctcccacaac gaggactaca 3841 ccatcgtgga gcagtacgag cgcaccgagg gccgccacca cctgttcctg tagtaaaccg 3901 gtgattcgtc agtagggttg taaaggtttt tcttttcctg agaaaacaac cttttgtttt 3961 ctcaggtttt gctttttggc ctttccctag ctttaaaaaa aaaaaagcaa aactcaccga 4021 ggcagttcca taggatggca agatcctggt attggtctgc gagttcactg ccgtataggc 4081 agctaagaaa tagtcgcgtg tagcgaagca gttttagagc tagaaatagc aagttaaaat 4141 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttcgttcac 4201 tgccgtatag gcagctaaga aacaaacagg aatcgaatgc aaccggcgca ggaacactgc 4261 cagcgcatca accccgggat gctagctaag cgggactctg gggttcgaaa tgaccgacca 4321 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt 4381 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 4441 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc 4501 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc 4561 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag 4621 acgccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc 4681 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagc 4741 tcgagtatgt cgaccgatgc ccttgagagc cttcaaccca g //