LOCUS Exported 9465 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses PbuCas13b from a SUMO-tagged bacterial expression construct. ACCESSION . VERSION . KEYWORDS pC0059 PbuCas13 (B03) His6-TwinStrep-SUMO-BsaI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9465) AUTHORS Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F TITLE Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. JOURNAL Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15. PUBMED 29449508 REFERENCE 2 (bases 1 to 9465) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9465 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1..18 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 28..45 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 58..141 /codon_start=1 /product="two Strep-Tag(R)II moieties connected by a linker for enhanced binding to Strep-Tactin(R), an engineered form of streptavidin (Schmidt et al., 2013)" /label=Twin-Strep-tag /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK" CDS 142..432 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" primer_bind complement(3867..3885) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3881..3928 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(4103..4131) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(4158..4176) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4244..4348 /gene="bla" /label=AmpR promoter CDS 4349..5209 /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(4567..4586) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5380..5968 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5869..5888 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6122..6139 /label=L4440 /note="L4440 vector, forward primer" misc_feature 6154..6293 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(6219..6238) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6379..6401 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6395..6586) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(7898..8980) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 8942..8961 /label=LacI-R /note="LacI, reverse primer" promoter complement(8981..9058) /gene="lacI" /label=lacI promoter primer_bind 9264..9283 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 9367..9386 /label=T7 /note="T7 promoter, forward primer" promoter 9367..9385 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 9386..9410 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." RBS 9425..9447 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" ORIGIN 1 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggctagctgg 61 agccatccgc agtttgaaaa aggtggtggt agcggtggtg gttcaggtgg tagtgcatgg 121 tcacaccctc agtttgagaa aatgtcggac tcagaagtca atcaagaagc taagccagag 181 gtcaagccag aagtcaagcc tgagactcac atcaatttaa aggtgtccga tggatcttca 241 gagatcttct tcaagatcaa aaagaccact cctttaagaa ggctgatgga agcgttcgct 301 aaaagacagg gtaaggaaat ggactcctta agattcttgt acgacggtat tagaatccaa 361 gctgatcaga cccctgaaga tttggacatg gaggataacg atattattga ggctcacaga 421 gaacagattg gtggatctat gcagaagcag gacaagctgt tcgtggatag gaagaagaac 481 gccatcttcg cctttcccaa gtacatcacc atcatggaga ataaggagaa gcccgagcct 541 atctactatg agctgacaga caagcacttc tgggccgcct ttctgaacct ggcccgccac 601 aacgtgtata ccacaatcaa ccacatcaat cggagactgg agatcgccga gctgaaggac 661 gatggctata tgatgggcat caagggcagc tggaacgagc aggccaagaa gctggacaag 721 aaggtgcggc tgagagatct gatcatgaag cacttccctt ttctggaggc cgccgcctac 781 gagatgacca atagcaagtc cccaaacaat aaggagcaga gggagaagga gcagtctgag 841 gccctgagcc tgaacaatct gaagaacgtg ctgttcatct ttctggagaa gctgcaggtg 901 ctgcgcaatt actatagcca ctacaagtat tctgaggaga gcccaaagcc catcttcgag 961 acatccctgc tgaagaacat gtataaggtg tttgacgcca atgtgaggct ggtgaagcgc 1021 gattatatgc accacgagaa catcgacatg cagagagatt tcacccacct gaatcggaag 1081 aagcaagtgg gcagaacaaa gaacatcatc gactccccta atttccacta ccactttgcc 1141 gataaggagg gcaacatgac catcgccggc ctgctgttct tcgtgagcct gttcctggac 1201 aagaaggatg ccatctggat gcagaagaag ctgaagggct tcaaggatgg caggaacctg 1261 cgcgagcaga tgaccaatga ggtgttttgc aggtcccgca tctctctgcc aaagctgaag 1321 ctggagaacg tgcagacaaa ggactggatg cagctggata tgctgaatga gctggtgagg 1381 tgtcccaaga gcctgtacga gcggctgaga gagaaggacc gcgagtcttt caaggtgcct 1441 ttcgatatct ttagcgacga ttataacgcc gaggaggagc cctttaagaa taccctggtg 1501 cggcaccagg acagattccc ttactttgtg ctgaggtatt tcgatctgaa cgagatcttc 1561 gagcagctgc gctttcagat cgatctgggc acctaccact tttccatcta taataagagg 1621 atcggcgacg aggatgaggt gcgccacctg acacaccacc tgtacggctt cgccagaatc 1681 caggactttg ccccccagaa ccagtctgag gagtggagaa agctggtgaa ggacctggat 1741 cacttcgaga caagccagga gccctacatc agcaagacag cccctcacta tcacctggag 1801 aatgagaaga tcggcatcaa gttctgcagc gcccacaaca atctgtttcc ctccctgcag 1861 accgacaaga catgtaacgg ccggtccaag ttcaatctgg gcacccagtt cacagccgag 1921 gcctttctgt ctgtgcacga gctgctgcct atgatgtttt actatctgct gctgaccaag 1981 gactactccc ggaaggagtc tgccgataag gtggagggca tcatcagaaa ggagatctct 2041 aacatctacg ccatctatga cgccttcgcc aacaatgaga tcaatagcat cgccgatctg 2101 accaggcgcc tgcagaacac aaatatcctg cagggccacc tgccaaagca gatgatcagc 2161 atcctgaagg gcaggcagaa ggatatgggc aaggaggccg agcgcaagat cggcgagatg 2221 atcgacgata cccagcggag actggacctg ctgtgcaagc agacaaacca gaagatcagg 2281 atcggcaagc gcaatgccgg cctgctgaag tccggcaaga tcgccgactg gctggtgaac 2341 gatatgatgc ggttccagcc tgtgcagaag gatcagaaca atatcccaat caacaatagc 2401 aaggccaact ccaccgagta ccggatgctg cagagagccc tggccctgtt cggctccgag 2461 aacttccggc tgaaggccta cttcaaccag atgaatctgg tgggcaacga caatcctcac 2521 ccatttctgg ccgagacaca gtgggagcac cagacaaaca tcctgtcttt ctacaggaat 2581 tatctggagg cccgcaagaa gtacctgaag ggcctgaagc cccagaactg gaagcagtat 2641 cagcactttc tgatcctgaa ggtgcagaaa accaacagga ataccctggt gacaggctgg 2701 aagaacagct tcaatctgcc acggggcatc tttacacagc ccatcagaga gtggttcgag 2761 aagcacaaca attccaagcg gatctacgac cagatcctgt ctttcgatag agtgggcttt 2821 gtggccaagg ccatccctct gtactttgcc gaggagtata aggacaacgt gcagcctttc 2881 tacgattatc ccttcaacat cggcaatcgg ctgaagccta agaagagaca gttcctggat 2941 aagaaggagc gcgtggagct gtggcagaag aacaaggagc tgtttaagaa ttacccatct 3001 gagaagaaga aaaccgacct ggcctatctg gatttcctga gctggaagaa gtttgagcgg 3061 gagctgagac tgatcaagaa ccaggacatc gtgacctggc tgatgttcaa ggagctgttt 3121 aatatggcca cagtggaggg cctgaagatc ggcgagatcc acctgcggga catcgatacc 3181 aacacagcca atgaggagtc caacaatatc ctgaacagaa tcatgccaat gaagctgccc 3241 gtgaaaacct acgagacaga caacaagggc aatatcctga aggagcggcc actggccacc 3301 ttctatatcg aggagacaga gacaaaggtg ctgaagcagg gcaactttaa ggccctggtg 3361 aaggacaggc gcctgaatgg cctgttctct tttgccgaga caacagatct gaatctggag 3421 gagcacccca tcagcaagct gtccgtggac ctggagctga tcaagtacca gaccacaaga 3481 atcagcatct tcgagatgac cctgggcctg gagaagaagc tgatcgacaa gtattccacc 3541 ctgccaacag attcttttag gaacatgctg gagcggtggc tgcagtgtaa ggccaatagg 3601 cccgagctga agaactacgt gaatagcctg atcgccgtgc gcaacgcctt ctcccacaat 3661 cagtacccta tgtatgatgc cacactgttt gccgaggtga agaagttcac cctgtttcca 3721 agcgtggaca caaagaagat cgagctgaac atcgcccccc agctgctgga gatcgtgggc 3781 aaggccatca aggagatcga gaagtccgag aacaagaatt aaggccgcac tcgaggcccg 3841 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 3901 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatatcccgc 3961 aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg 4021 ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa 4081 tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa 4141 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4201 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4261 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4321 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4381 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4441 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4501 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 4561 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 4621 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 4681 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 4741 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 4801 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 4861 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 4921 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 4981 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5041 taaatctgga gccggtgagc gtgggtcacg cggtatcatt gcagcactgg ggccagatgg 5101 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5161 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5221 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5281 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5341 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5401 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5461 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 5521 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 5581 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 5641 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 5701 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 5761 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 5821 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 5881 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 5941 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6001 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 6061 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 6121 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 6181 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca 6241 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac 6301 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 6361 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 6421 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt 6481 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc 6541 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt 6601 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga 6661 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg 6721 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta 6781 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca 6841 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga 6901 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct 6961 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc 7021 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc 7081 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt 7141 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt 7201 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg 7261 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca 7321 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag 7381 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct 7441 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat 7501 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc 7561 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac 7621 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt 7681 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc 7741 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc 7801 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc 7861 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg 7921 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 7981 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 8041 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 8101 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 8161 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 8221 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 8281 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 8341 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 8401 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 8461 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 8521 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 8581 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 8641 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 8701 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 8761 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 8821 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 8881 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 8941 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac 9001 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg 9061 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg 9121 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt 9181 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 9241 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 9301 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 9361 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 9421 taattttgtt taactttaag aaggagatat accatgggca gcagc //