LOCUS Exported 9989 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for Cas13a. ACCESSION . VERSION . KEYWORDS p2CT-His-MBP-Ere_Cas13a_WT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9989) AUTHORS East-Seletsky A, O'Connell MR, Burstein D, Knott GJ, Doudna JA TITLE RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. JOURNAL Mol Cell. 2017 May 4;66(3):373-383.e3. doi: 10.1016/j.molcel.2017.04.008. PUBMED 28475872 REFERENCE 2 (bases 1 to 9989) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9989 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 81..669 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 570..589 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 823..840 /label=L4440 /note="L4440 vector, forward primer" misc_feature 855..994 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(920..939) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1080..1102 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(1096..1287) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 2745..2764 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 2848..2867 /label=T7 /note="T7 promoter, forward primer" promoter 2848..2866 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 2955..2977 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2997..3014 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 3024..4124 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" primer_bind 4098..4121 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 4179..4199 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(8366..8384) /label=T7 Term /note="T7 terminator, reverse primer" terminator 8380..8427 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(8793..8821) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(8848..8866) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8934..9038 /gene="bla" /label=AmpR promoter CDS 9039..9899 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(9257..9276) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 61 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 121 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 181 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 241 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 301 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 361 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 421 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 481 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 541 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 601 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 661 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 721 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 781 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 841 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 901 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 961 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 1021 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 1081 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg 1141 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc 1201 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt 1261 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat 1321 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga 1381 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga 1441 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc 1501 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga 1561 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca 1621 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt 1681 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat 1741 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga 1801 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg 1861 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct 1921 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca 1981 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca 2041 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc 2101 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac 2161 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct 2221 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg 2281 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg 2341 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg 2401 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag 2461 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact 2521 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg 2581 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc 2641 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa 2701 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata 2761 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag 2821 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt 2881 ttccctctag tgccggctcc ggagagctct ttaattaagc ggccgccctg caggactcga 2941 gttctagaaa taattttgtt taactttaag aaggagatat acatatgaaa tcttctcacc 3001 atcaccatca ccatggttct tctatgaaaa tcgaagaagg taaactggta atctggatta 3061 acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 3121 gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 3181 caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 3241 aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt 3301 ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 3361 cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga 3421 tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 3481 aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 3541 aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 3601 tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 3661 tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 3721 ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 3781 gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 3841 acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 3901 cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 3961 cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga 4021 acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 4081 gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca 4141 acaacaacaa taacaataac aacaacctcg ggatcgagga aaacctgtac ttccaatcca 4201 atgcaatgct gcgtcgtgat aaggaagtga aaaagcttta taacgttttt aatcagatcc 4261 aggtcggtac gaagcctaaa aaatggaata atgatgaaaa attgtcgccc gaagagaatg 4321 aacgtcgcgc tcaacagaaa aatatcaaga tgaaaaacta caaatggcgt gaagcctgct 4381 caaagtatgt tgagtcctcg caacgcatta tcaacgatgt tattttttac tcttatcgta 4441 aagcgaaaaa taagttacgc tacatgcgca agaatgagga tatcttgaag aagatgcaag 4501 aggcagagaa gttgtccaag ttcagtggcg gcaagttgga ggattttgtc gcatacacct 4561 tgcgtaagtc gttagtagtc tccaagtacg atacacaaga gttcgatagt ttagctgcta 4621 tggttgtatt ccttgagtgt attggtaaaa ataacatctc cgatcatgag cgcgagatcg 4681 tttgtaaact gcttgaatta attcgtaagg actttagtaa gttggatcca aatgttaagg 4741 ggtcacaagg tgcaaacatt gtccgcagtg tccgcaatca gaatatgatt gtacaacccc 4801 agggggatcg tttccttttc ccccaggtgt atgcgaagga gaatgagacc gtcactaata 4861 aaaatgtaga aaaagaaggt ttaaacgagt tccttctgaa ttacgcgaat cttgacgacg 4921 agaaacgcgc ggaaagtttg cgcaaattgc gccgtattct tgacgtttac ttttctgcac 4981 caaaccacta tgaaaaggac atggatatca cattaagcga caacattgaa aaggagaagt 5041 tcaacgtgtg ggagaaacac gaatgtggca aaaaggagac aggcttattt gtggacatcc 5101 cagatgtatt aatggaagca gaagcagaga acattaaatt agacgccgtg gtagagaaac 5161 gtgagcgtaa agtcttaaac gatcgtgtac gtaaacaaaa catcatctgc taccgctata 5221 cgcgcgcagt ggtggagaaa tacaattcca acgagccttt gttctttgag aataatgcca 5281 tcaaccaata ctggatccac catattgaaa acgccgtcga acgtattctt aaaaactgta 5341 aagcaggtaa gttgttcaag ctgcgcaagg ggtatcttgc agagaaagtc tggaaagatg 5401 ccatcaactt aatctccatc aaatacattg cgttagggaa ggccgtatac aattttgcgt 5461 tagacgacat ctggaaggac aagaaaaata aggaactggg gatcgtggat gagcgcatcc 5521 gtaacggcat cactagtttt gattatgaaa tgatcaaggc gcacgagaac ttgcaacgtg 5581 aacttgctgt ggacattgcg ttttcggtga acaaccttgc acgcgctgtt tgtgacatga 5641 gtaacttggg taataaagaa tcggatttcc tgttgtggaa acgcaacgac attgccgaca 5701 agttaaagaa caaggatgac atggctagcg ttagcgctgt actgcagttt ttcggtggca 5761 agtccagctg ggatatcaac atttttaaag atgcctataa aggtaaaaag aaatacaatt 5821 acgaagttcg ttttatcgac gatctgcgca aggcgatcta ttgtgcacgc aacgagaatt 5881 tccatttcaa gacagcgttg gttaacgatg aaaagtggaa tactgaattg tttggcaaaa 5941 tcttcgaacg cgagacggag ttttgcctta atgtggagaa agaccgcttc tactcgaaca 6001 acttatacat gttctaccag gtaagtgaac tgcgcaacat gcttgatcac ctgtatagtc 6061 gctcagtaag tcgcgccgcc caagtgcctt cttacaattc tgtaattgtt cgcactgctt 6121 tccccgagta tatcacgaat gtattaggat atcagaaacc aagctacgat gctgacacgt 6181 tagggaagtg gtacagcgcc tgctactatt tgttgaaaga aatctattac aattccttct 6241 tacagtcgga tcgtgcgttg cagctttttg agaaatcagt taaaaccctg tcatgggacg 6301 ataaaaaaca acaacgtgcg gtagataatt ttaaggatca cttttccgac attaaatcag 6361 cctgcactag tttagcccag gtgtgtcaga tttatatgac ggagtacaac cagcagaaca 6421 accaaattaa aaaggttcgc tcatcaaatg actctatttt cgatcaacca gtctaccaac 6481 attacaaggt cttattgaag aaggcgattg caaacgcttt tgcggactat ttgaagaata 6541 acaaggactt attcggtttt atcggcaaac cgttcaaggc taatgaaatc cgtgagattg 6601 acaaagaaca gtttctgccc gactggacta gtcgcaaata cgaagcatta tgtatcgagg 6661 ttagcggaag ccaagaattg cagaaatggt acatcgtagg taaattcttg aacgcccgta 6721 gtttaaactt gatggtgggt tccatgcgct cctatatcca atacgtcaca gatattaaac 6781 gccgtgcagc atctattggt aatgaattgc atgtatcggt acatgatgta gaaaaggtag 6841 agaagtgggt gcaggtcatc gaggtttgtt ccttactggc ctcgcgtact agtaatcagt 6901 tcgaagacta ctttaacgat aaggatgact acgcacgtta cttaaagtca tacgtcgatt 6961 tttcaaacgt tgacatgccc tccgagtact cagccttagt ggattttagc aatgaggagc 7021 aatctgattt gtatgtggac ccaaagaacc ctaaggtaaa tcgcaacatc gtgcactcga 7081 aactgtttgc agcagatcat attttacgcg acatcgtcga gccagtcagc aaggataata 7141 ttgaagaatt ttactcacag aaggcggaga ttgcttattg taaaatcaag gggaaagaga 7201 tcacggcgga ggagcaaaaa gccgtgttga aatatcaaaa actgaaaaac cgtgtggaac 7261 tgcgtgatat cgtagagtat ggcgaaatta tcaacgagtt gttgggacag ctgattaatt 7321 ggagcttcat gcgtgaacgt gacttgttat acttccaact tggatttcat tacgactgtt 7381 tacgcaacga tagtaagaaa cctgaaggtt ataagaatat taaagtggac gagaattcta 7441 tcaaggacgc tatcttatac cagatcattg gaatgtatgt caatggggta actgtatacg 7501 cgcctgaaaa agatggggat aaattgaagg aacagtgtgt gaaaggagga gtgggagtga 7561 aagtaagtgc gttccaccgc tattctaaat atttaggact gaatgaaaaa acgctttaca 7621 acgccggtct ggagattttc gaagtcgtag cggagcatga ggacattatc aacttacgca 7681 acggcatcga tcactttaag tactatttgg gcgattatcg ttctatgttg tcgatttaca 7741 gcgaagtctt cgatcgcttt ttcacttatg acattaaata ccagaaaaat gtattgaatt 7801 tactgcaaaa tatccttttg cgccacaatg tcatcgtcga gccgattttg gagtcgggtt 7861 tcaagaccat tggggagcag actaaaccag gtgcaaagtt gtcgattcgc agtattaaaa 7921 gtgacacatt ccaatacaag gttaaaggcg ggacattgat tacagacgct aaggacgagc 7981 gttaccttga aacgattcgt aagattctgt actacgctga aaatgaagag gataacctga 8041 aaaagtcagt ggtcgttaca aacgctgaca aatacgagaa aaataaggag tcagacgacc 8101 agaataaaca gaaagagaaa aagaataagg acaataaggg gaaaaagaac gaggagacaa 8161 aatctgacgc cgagaaaaac aataacgaac gcttatcata caaccccttt gcgaatctta 8221 actttaagct gagcaactaa taacattgga agtggataac ggatccgcga tcgcggcgcg 8281 ccacctggtg gccggccggt accacgcgtg cgcgctgatc cggctgctaa caaagcccga 8341 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 8401 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatccacag 8461 gacgggtgtg gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag 8521 gactgggcgg cggccaaagc ggtcggacag tgctccgaga acgggtgcgc atagaaattg 8581 catcaacgca tatagcgcta gcagcacgcc atagtgactg gcgatgctgt cggaatggac 8641 gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag 8701 ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact 8761 gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca 8821 aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 8881 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 8941 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 9001 cctgataaat gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg 9061 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 9121 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 9181 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 9241 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc 9301 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 9361 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 9421 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 9481 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 9541 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt 9601 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 9661 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 9721 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 9781 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 9841 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 9901 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 9961 aaaggatcta ggtgaagatc ctttttgat //