LOCUS Exported 8532 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression vector for xCas9(3.7)-BE3. ACCESSION . VERSION . KEYWORDS xCas9(3.7)-BE3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8532) AUTHORS Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR TITLE Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. JOURNAL Nature. 2018 Feb 28. pii: nature26155. doi: 10.1038/nature26155. PUBMED 29512652 REFERENCE 2 (bases 1 to 8532) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8532 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 74..453 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 454..657 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 608..628 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 699..718 /label=T7 /note="T7 promoter, forward primer" promoter 699..717 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 731..1417 /codon_start=1 /product="cytidine deaminase (C to U editing enzyme) from rat" /label=APOBEC-1 /note="can use ssDNA as a substrate (Komor et al., 2016)" /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI LWATGLK" CDS 1466..5566 /codon_start=1 /product="Streptococcus pyogenes Cas9 mutant with broad PAM compatibility (Hu et al., 2018)" /label=xCas9-3.7 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5579..5827 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5840..5860 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5869..5886 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5909..5926) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5915..6139 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6210..6226) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6210..6226) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6223..6245) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6234..6250 /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)." promoter complement(6258..6288) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6303..6324 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6441..6458) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6612..7200) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6692..6711) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7371..8231) /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 7994..8013 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8232..8336) /gene="bla" /label=AmpR promoter primer_bind complement(8415..8434) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc 61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca 121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt 661 ttagtgaacc gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag 721 agccgccacc atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg 781 gatcgagccc catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg 841 cctgctttac gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa 901 cactaacaag cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg 961 tccgaacaca aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag 1021 tagggccatc actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc 1081 aaggctgtac caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc 1141 aggtgtgact atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt 1201 gaattatagc ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact 1261 gtacgttctt gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag 1321 aaggaagcag ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg 1381 actgccccca cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac 1441 ctcagagtcc gccacacccg aaagtgacaa gaagtactcc attgggctcg ctatcggcac 1501 aaacagcgtc ggctgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa 1561 agttctgggc aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt 1621 cgactccggg gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac 1681 ccgcagaaag aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt 1741 ggatgactct ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca 1801 cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc 1861 aaccatatat catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt 1921 gatctatctc gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga 1981 cctgaaccca gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa 2041 tcagcttttc gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag 2101 cgctaggctg tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa 2161 gaagaacggc ctgtttggta atcttatcgc cctgtccctc gggctgaccc ccaactttaa 2221 atctaacttc gacctggccg aagataccaa gcttcaactg agcaaagaca cctacgatga 2281 tgatctcgac aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc 2341 aaagaacctg tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac 2401 caaagctccg ctgagcgcta gtatgatcaa gctctatgat gagcaccacc aagacttgac 2461 tttgctgaag gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga 2521 tcagtctaaa aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta 2581 caaatttatt aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct 2641 taacagagaa gatctgttgc gcaaacagcg cactttcgac aatggaatca tcccccacca 2701 gattcacctg ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt 2761 gaaagataac agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg 2821 ccccctcgcc cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat 2881 cactccctgg aacttcgaga aagtcgtgga taagggggcc tctgcccagt ccttcatcga 2941 aaggatgact aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct 3001 gctgtacgag tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg 3061 gatgagaaag ccagcattcc tgtctggaga tcagaagaaa gctattgtgg acctcctctt 3121 caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga 3181 atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac 3241 gtatcacgat ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga 3301 ggacattctt gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga 3361 agaacgcttg aaaacttacg ctcatctctt cgacgacaaa gtcatgaagc agctcaagag 3421 gcgccgatat acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa 3481 gcagagtgga aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt 3541 cattcagttg atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt 3601 ttctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat 3661 caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag 3721 gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacca cccagaaggg 3781 acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc 3841 ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct 3901 gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct 3961 ctccgactac gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga 4021 taataaagtg ttgacaagat ccgataaaaa cagagggaag agtgataacg tcccctcaga 4081 agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac 4141 acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa 4201 agccggtttc atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca 4261 aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt 4321 gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta 4381 taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt 4441 aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta 4501 taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac 4561 cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc 4621 caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt 4681 gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa 4741 catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa 4801 aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg 4861 attcgattct cctacagtcg cttacagtgt actggttgtg gctaaagtgg agaaagggaa 4921 gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag 4981 cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga 5041 cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat 5101 gctcgctagt gcgggcgtgc tgcagaaagg taacgagctg gcactgccct ctaaatacgt 5161 taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga 5221 gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat 5281 aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc 5341 ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt 5401 tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacta ccatagacag 5461 aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac 5521 ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagactctg gtggttctac 5581 taatctgtca gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat 5641 cctcatgctc ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact 5701 cgtgcacacc gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc 5761 ccctgaatac aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa 5821 gatgctctct ggtggttctc ccaagaagaa gaggaaagtc taaccggtca tcatcaccat 5881 caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5941 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 6001 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 6061 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 6121 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg 6181 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 6241 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc 6301 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6361 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 6421 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 6481 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 6541 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6601 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6661 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6721 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6781 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6841 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6901 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6961 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 7021 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 7081 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 7141 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 7201 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7261 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 7321 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 7381 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 7441 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 7501 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 7561 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 7621 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7681 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 7741 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7801 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7861 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7921 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 7981 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 8041 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 8101 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 8161 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 8221 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 8281 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 8341 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta 8401 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct 8461 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa 8521 ggcttgaccg ac //