LOCUS Exported 10115 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for Cas13a. ACCESSION . VERSION . KEYWORDS p2CT-His-MBP-Cam_Cas13a_WT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10115) 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 10115) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10115 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(18..36) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 104..208 /gene="bla" /label=AmpR promoter CDS 209..1069 /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(427..446) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1240..1828 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1729..1748 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1982..1999 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2014..2156 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2082..2101) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2242..2264 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2258..2449) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 3907..3926 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4010..4029 /label=T7 /note="T7 promoter, forward primer" promoter 4010..4028 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 4117..4139 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4159..4176 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4186..5286 /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 5260..5283 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 5341..5361 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(9651..9669) /label=T7 Term /note="T7 terminator, reverse primer" terminator 9665..9712 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(10078..10106) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" ORIGIN 1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc 3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc 3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc 3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc 3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc 3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc 4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa 4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat 4141 atacatatga aatcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa 4201 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt 4261 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa 4321 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac 4381 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg 4441 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt 4501 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac 4561 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag 4621 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac 4681 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat 4741 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg 4801 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg 4861 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta 4921 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca 4981 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg 5041 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg 5101 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc 5161 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt 5221 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg 5281 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag 5341 gaaaacctgt acttccaatc caatgcaatg aagttcagta aagtcgatca tacacgtagt 5401 gctgttggta ttcaaaaggc cacagatagc gttcacggga tgctgtatac tgaccccaaa 5461 aaacaggaag tcaatgatct tgataaacgc ttcgaccaat taaacgtaaa ggcgaaacgc 5521 ttgtataacg tgttcaatca atccaaagcg gaggaggacg atgacgagaa acgtttcgga 5581 aaggtggtaa aaaaattaaa tcgcgaactg aaggatctgc tttttcaccg tgaagtgtct 5641 cgttataact ccattgggaa cgccaagtac aattactatg ggattaaatc taacccagaa 5701 gaaattgtct ccaacctggg aatggtggag agtcttaagg gcgagcgtga tccccaaaag 5761 gttatctcaa aattactgct ttattacctt cgtaagggac ttaagccggg aaccgacggc 5821 ctgcgtatga ttctggaggc ttcatgtggt ttacgtaaac tttccggtga cgaaaaggaa 5881 ttaaaggttt ttcttcaaac cctggatgag gatttcgaaa agaagacgtt taaaaaaaac 5941 ctgattcgct cgatcgaaaa tcaaaatatg gccgttcagc cttccaatga gggtgatcct 6001 atcatcggca ttacccaggg acgttttaac tcgcagaaga acgaagagaa aagcgccatc 6061 gaacgcatga tgtcaatgta tgctgacctg aatgaggatc accgtgaaga tgtattacgc 6121 aaactgcgtc gccttaatgt cctgtatttt aacgttgaca ccgaaaaaac agaggagccg 6181 acacttccag gcgaggtgga tacaaatccc gtttttgagg tgtggcatga ccacgaaaaa 6241 ggcaaagaaa atgaccgcca gtttgctacc tttgcaaaaa ttttaactga agaccgtgag 6301 acacgtaaaa aggagaagtt ggctgtcaaa gaggcgttga acgatttgaa atccgccatt 6361 cgcgaccata atattatggc ctaccgttgc tctattaagg ttactgaaca ggacaaagac 6421 gggctgttct tcgaggatca acgtatcaat cgcttttgga tccaccacat cgagagcgca 6481 gtcgagcgca tcttagcatc gattaatccc gaaaaattgt ataaactgcg cattgggtat 6541 cttggagaga aggtatggaa ggatcttttg aactatcttt ctattaaata catcgccgtg 6601 ggcaaggcag tgtttcactt cgctatggag gatttgggaa aaactgggca agacatcgaa 6661 ttgggcaaac tgtcaaacag tgtaagcggc ggcttaacat ccttcgatta cgagcagatt 6721 cgcgctgacg aaacgcttca acgtcagtta agcgttgaag ttgcctttgc agcaaataac 6781 ctgttccgtg ccgttgtagg acagaccggc aagaagattg agcaatccaa atctgaggag 6841 aatgaggagg acttcctttt atggaaagct gaaaaaattg cggaatcaat taaaaaagaa 6901 ggtgagggaa acacgcttaa aagtatcttg caattcttcg ggggagcgag ctcctgggat 6961 ttgaaccatt tttgcgcagc gtatggtaac gagtcgtctg cattagggta tgaaactaaa 7021 ttcgcagacg acctgcgcaa ggcgatttac tcactgcgta atgagacctt tcattttact 7081 acacttaaca aaggatcatt tgactggaat gcaaagttaa tcggtgacat gtttagccac 7141 gaggctgcaa cggggatcgc agtagaacgc acgcgctttt actcgaacaa tttaccgatg 7201 ttttaccgcg agtcggactt gaaacgcatc atggaccatt tatacaatac ctaccacccc 7261 cgtgcgtccc aagttcctag ttttaactca gttttcgtgc gcaagaattt ccgcctgttt 7321 ctgagtaata cgctgaacac caatacctca ttcgacactg aagtatacca aaagtgggag 7381 agcggtgtct attatctgtt caaggagatc tattataaca gtttcttacc aagtggtgac 7441 gcccaccatc ttttctttga agggttacgt cgcatccgta aagaagctga caatctgccg 7501 atcgtgggta aagaagctaa gaaacgcaat gcggttcaag attttggtcg tcgttgtgac 7561 gaacttaaga atctttctct gagcgccatt tgccagatga ttatgaccga atataatgag 7621 caaaataatg gaaatcgtaa ggtcaagtct acacgtgaag ataaacgtaa gccagatatt 7681 ttccagcact ataaaatgct gttactgcgc accttgcagg aggcttttgc gatctatatt 7741 cgtcgcgaag agttcaagtt tatcttcgac ttgccaaaaa ccctttatgt gatgaaacca 7801 gtcgaagagt tcttgcctaa ttggaaatcc gggatgttcg atagtttagt ggaacgcgta 7861 aagcagtcgc ccgacttaca gcgctggtac gtgttgtgta aatttttaaa tgggcgtttg 7921 ttgaatcaat tatcaggggt aattcgctca tatatccagt ttgcaggaga tatccaacgc 7981 cgcgcaaaag ctaatcataa ccgcttgtat atggataaca cgcagcgcgt tgagtattat 8041 tcaaacgtct tagaagtggt ggatttctgt attaagggca cttctcgctt ctccaacgtg 8101 ttctctgatt atttccgcga cgaagatgca tatgccgatt accttgacaa ttacttgcaa 8161 tttaaagatg agaaaatcgc ggaggtctcc agttttgcag ccttgaagac attttgtaac 8221 gaagaggagg ttaaagcagg aatttacatg gatggagaaa accccgttat gcagcgcaat 8281 atcgttatgg ccaaactttt cgggccggat gaggttctga aaaatgtggt tccgaaggtt 8341 acgcgtgagg agatcgagga atattaccag ttagagaaac agattgctcc atatcgccag 8401 aatggatatt gtaaatccga ggaggaccaa aagaaacttc ttcgctttca gcgtatcaaa 8461 aatcgtgttg aattccagac gatcaccgag ttcagcgaga ttattaatga gctgttagga 8521 cagctgattt cctggagctt cttacgtgaa cgcgacttgc tgtactttca gctgggcttt 8581 cactaccttt gtcttcacaa tgacacggag aagcctgccg aatacaagga aatttctcgc 8641 gaggacggca cagttatccg taatgccatt ttgcatcagg tggctgctat gtatgtgggt 8701 ggattgcccg tttacacctt agccgacaag aagttggccg cattcgagaa gggcgaggcg 8761 gattgcaaac tgagcatttc gaaagataca gcaggggccg gtaagaagat taaggatttt 8821 ttccgctaca gtaaatacgt attgattaaa gatcgtatgc tgaccgacca gaatcagaaa 8881 tacacaatct acttagcggg tctggagttg ttcgagaaca cggatgaaca tgacaatatt 8941 acggacgtac gcaagtacgt agatcatttt aagtattatg ctacgagtga tgaaaacgcc 9001 atgtctattc ttgatctgta cagtgagatt catgatcgtt tcttcacgta tgacatgaag 9061 tatcaaaaga acgtagccaa catgcttgag aatatccttt tacgtcactt cgtgcttatc 9121 cgtcctgagt ttttcactgg tagtaagaaa gtcggggaag gaaaaaagat tacatgtaag 9181 gctcgtgcac aaattgagat tgcagaaaac gggatgcgtt cagaggactt tacgtataaa 9241 ctgtccgatg ggaaaaagaa cattagcacc tgtatgattg cggcacgcga ccagaagtat 9301 ctgaatacgg tcgcgcgttt actgtactat ccacacgagg caaagaagtc gatcgtcgac 9361 acgcgcgaaa agaaaaacaa taagaagaca aaccgcggtg acggcacctt caataagcag 9421 aaaggtactg cacgtaagga aaaggataat gggccccgcg aattcaacga tacgggcttt 9481 agcaatacgc catttgcagg tttcgacccc tttcgtaata gctaataaca ttggaagtgg 9541 ataacggatc cgcgatcgcg gcgcgccacc tggtggccgg ccggtaccac gcgtgcgcgc 9601 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca 9661 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg 9721 aggaactata tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag 9781 tggctccaag tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg gacagtgctc 9841 cgagaacggg tgcgcataga aattgcatca acgcatatag cgctagcagc acgccatagt 9901 gactggcgat gctgtcggaa tggacgatat cccgcaagag gcccggcagt accggcataa 9961 ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt 10021 tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt 10081 aaagcttatc gatgataagc tgtcaaacat gagaa //