LOCUS Exported 9422 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for Cas13a. ACCESSION . VERSION . KEYWORDS p2CT-His-MBP-Ppr_Cas13a_WT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9422) 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 9422) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9422 /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(8958..8976) /label=T7 Term /note="T7 terminator, reverse primer" terminator 8972..9019 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(9385..9413) /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 cgtgtgtcga aagtgaaagt taaagatggg 5401 ggcaaagaca agatggtctt ggtccatcgt aagacgaccg gtgcccaact tgtttattca 5461 gggcagcccg ttagcaacga gacgtctaat attctgccag aaaaaaagcg ccagtcattc 5521 gaccttagca ccctgaacaa aacaattatc aaattcgata ctgcgaaaaa gcaaaagtta 5581 aatgtggacc agtataagat cgtagaaaag atcttcaagt atcccaagca ggagctgccc 5641 aagcaaatta aagcagagga gatcctgccg tttttgaatc acaaattcca ggagccagtc 5701 aagtattgga aaaacgggaa ggaagagagc ttcaatttaa cattattgat cgtcgaggca 5761 gtccaagcac aggacaagcg taaacttcag ccctattatg actggaagac ctggtacatc 5821 caaactaaaa gcgatttact gaagaagagt atcgagaaca atcgtatcga tttgactgag 5881 aacttgtcaa agcgcaagaa ggcattgttg gcctgggaga cagagttcac agcatccggt 5941 tcaatcgacc tgacgcatta ccataaggtc tatatgacgg atgttctgtg caaaatgtta 6001 caagatgtta agccgctgac agatgataaa gggaagatca acaccaatgc gtatcatcgt 6061 gggcttaaga aagccctgca gaaccaccaa ccagccatct ttggaacacg cgaggtgcct 6121 aatgaggcta atcgtgcaga caatcagttg agcatttatc atttggaggt cgtgaagtat 6181 cttgagcact attttccaat taaaacttcg aaacgccgca ataccgccga cgatatcgct 6241 cattatttaa aggcacagac gttgaagaca acgattgaaa agcagctggt aaacgccatc 6301 cgcgcgaaca tcatccaaca aggcaaaacc aatcatcacg agttgaaagc ggacacaaca 6361 tcgaatgatt tgatccgtat taaaactaac gaagccttcg tcttaaactt aactggtacc 6421 tgtgcgtttg cagcgaacaa tattcgcaac atggtggaca acgagcaaac taacgacatc 6481 ctgggaaagg gggactttat caagagctta ctgaaggaca atacgaacag ccaattgtac 6541 tcgttttttt ttggtgaagg attatccacg aataaggcag agaaagagac gcaactttgg 6601 ggtatccgtg gggctgttca acagatccgc aataatgtaa atcattacaa gaaagacgcc 6661 cttaaaaccg tgttcaacat ctcaaatttc gagaatccca ccattactga cccgaaacaa 6721 cagactaact atgcagacac catctataaa gcgcgcttca tcaatgaatt ggaaaagatc 6781 cctgaagcct ttgcacagca gcttaaaact ggcggtgctg tctcctatta cactatcgaa 6841 aacctgaaat cattattgac tacgtttcag ttttcgctgt gtcgtagcac gattccattt 6901 gcacctggtt tcaaaaaggt ttttaatgga ggaatcaatt accaaaacgc caagcaagat 6961 gaatctttct atgagcttat gctggaacaa tacttgcgta aagagaattt cgccgaagag 7021 agctacaatg cacgttattt tatgctgaag ttgatctata acaatctttt cctgccgggc 7081 tttacgaccg accgtaaggc gttcgccgac agtgtgggat ttgtgcaaat gcagaacaaa 7141 aagcaagctg agaaagtcaa ccctcgcaag aaagaggcgt atgcgttcga ggcggtacgt 7201 ccgatgaccg cagcagactc tatcgcagat tacatggcat acgttcaaag tgaattaatg 7261 caggagcaaa acaaaaagga ggaaaaagtt gccgaggaaa ctcgcatcaa ttttgaaaag 7321 ttcgtccttc aggtgttcat taaagggttc gattcgttct tgcgcgccaa agagtttgac 7381 ttcgtccaaa tgccacaacc tcaattgact gctacggcgt ccaaccaaca aaaagccgac 7441 aagttaaacc aactggaagc ctctattact gctgattgca aacttacgcc ccaatatgcg 7501 aaagcggacg atgctacaca tatcgcgttc tacgtctttt gcaaattact ggatgcggca 7561 cacttgtcta atttgcgtaa cgagctgatt aagtttcgcg aatccgttaa cgaattcaaa 7621 ttccaccatt tgctggaaat cattgagatt tgccttttat ctgcagatgt tgtgccaaca 7681 gactatcgtg atctttatag tagcgaagct gactgcttag cacgtctgcg cccgttcatt 7741 gaacagggcg cagacatcac aaactggtca gacttatttg tacaatctga caaacacagc 7801 ccagtcatcc acgcaaacat tgagcttagt gttaaatatg gcacaactaa actgttagag 7861 cagattatca ataaagatac tcaattcaag accacagagg caaattttac cgcctggaat 7921 actgcgcaga agagcatcga acagcttatt aagcaacgcg aggaccatca cgagcaatgg 7981 gtcaaggcta aaaacgccga cgacaaagaa aaacaagagc gcaaacgcga gaagtccaac 8041 tttgcgcaaa agtttattga gaaacacggt gacgattatc ttgatatctg cgattatatc 8101 aacacttata actggcttga caataaaatg cacttcgtgc acctgaaccg tctgcacggc 8161 ttgactatcg agcttttagg tcgcatggcc gggtttgtcg ccctgtttga tcgcgatttt 8221 caatttttcg atgagcaaca gatcgctgac gagttcaagt tacacggatt tgtgaactta 8281 cacagtattg acaagaagct caatgaagtc cctactaaaa agatcaaaga gatctacgac 8341 attcgtaaca aaatcattca aattaacgga aacaagatca atgagtccgt gcgtgccaac 8401 ttaattcaat ttatctcgtc taaacgtaat tactataata acgccttctt gcacgtgtcc 8461 aatgatgaga tcaaagaaaa gcagatgtat gatatccgca accatatcgc gcacttcaac 8521 tatttgacga aggacgcggc tgacttctct ttgattgact tgattaacga gcttcgcgaa 8581 ttgttacatt atgatcgcaa acttaagaac gctgtttcca aggcctttat cgacctgttt 8641 gataagcacg gcatgattct gaagcttaag cttaacgcag accacaagtt aaaggttgag 8701 tccctggagc caaagaaaat ttaccatctg ggatcatctg cgaaggacaa gccggagtat 8761 caatattgca ccaaccaagt catgatggca tactgtaaca tgtgccgctc ccttttagag 8821 atgaaaaaat aataacattg gaagtggata acggatccgc gatcgcggcg cgccacctgg 8881 tggccggccg gtaccacgcg tgcgcgctga tccggctgct aacaaagccc gaaaggaagc 8941 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg 9001 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg 9061 tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc 9121 ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg 9181 catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc 9241 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg 9301 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc 9361 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag 9421 aa //