LOCUS Exported 8451 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CMV and T7 promoter expression plasmid for human codon optimized SpRY-HF1(SpRY=A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/ R1333P/R1335Q/T1337R;HF1=N497A/R661A/Q695A/Q926A) with a c-terminal bi-partite NLS, 3x flag tag, and P2A-EGFP. ACCESSION . VERSION . KEYWORDS pCMV-T7-SpRY-HF1-P2A-EGFP (RTW5008) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8451) AUTHORS Walton RT, Christie KA, Whittaker MN, Kleinstiver BP TITLE Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. JOURNAL Science. 2020 Apr 17;368(6488):290-296. doi: 10.1126/science.aba8853. Epub 2020 Mar 26. PUBMED 32217751 REFERENCE 2 (bases 1 to 8451) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8451 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 403..412 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 409..2127 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 4567..4587 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4600..4665 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4684..4740 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 4741..5457 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4786..4807) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5047..5066) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5394..5415 /label=EGFP-C /note="EGFP, forward primer" CDS 5466..5483 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5506..5523) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5512..5736 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5807..5823) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5807..5823) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5820..5842) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5831..5847 /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(5855..5885) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5900..5921 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6038..6055) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6209..6797) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6289..6308) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6968..7828) /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 7591..7610 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7829..7933) /gene="bla" /label=AmpR promoter primer_bind complement(8012..8031) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 8203..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggacaagaag 421 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 481 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 541 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgagag aacccggctg 601 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 661 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 721 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 781 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 841 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 901 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 961 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 1021 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 1081 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 1141 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 1201 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 1261 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 1321 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 1381 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 1441 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 1501 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 1561 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 1621 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 1681 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 1741 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 1801 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 1861 ggcgcttccg cccagagctt catcgagcgg atgaccgcct tcgataagaa cctgcccaac 1921 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 1981 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 2041 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 2101 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 2161 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 2221 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 2281 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 2341 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcgc cctgagccgg 2401 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 2461 tccgacggct tcgccaacag aaacttcatg gccctgatcc acgacgacag cctgaccttt 2521 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 2581 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 2641 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 2701 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 2761 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 2821 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 2881 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 2941 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 3001 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 3061 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 3121 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 3181 cgggccatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 3241 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 3301 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 3361 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 3421 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 3481 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 3541 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 3601 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 3661 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 3721 ttcagcaaag agtctatcag acccaagagg aacagcgata agctgatcgc cagaaagaag 3781 gactgggacc ctaagaagta cggcggcttc ctgtggccca ccgtggccta ttctgtgctg 3841 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 3901 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 3961 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 4021 gagctggaaa acggccggaa gagaatgctg gcctctgcca agcagctgca gaagggaaac 4081 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 4141 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 4201 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 4261 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 4321 caggccgaga atatcatcca cctgtttacc ctgaccagac tgggagcccc tagagccttc 4381 aagtactttg acaccaccat cgaccccaag cagtacagaa gcaccaaaga ggtgctggac 4441 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 4501 ctgggaggcg acggatccgg cggaggcgga agcgggaaaa gaaccgccga cggcagcgaa 4561 ttcgagccca agaagaagag gaaagtctcg agcggaggcg actacaaaga ccatgacggt 4621 gattataaag atcatgacat cgattacaag gatgacgatg acaagggagg cggtggaagc 4681 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg 4741 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 4801 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 4861 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 4921 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 4981 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 5041 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 5101 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 5161 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 5221 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 5281 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 5341 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 5401 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga 5461 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt 5521 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5581 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 5641 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 5701 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 5761 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 5821 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 5881 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 5941 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 6001 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 6061 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 6121 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 6181 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 6241 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 6301 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 6361 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 6421 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 6481 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 6541 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 6601 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 6661 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 6721 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 6781 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 6841 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 6901 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 6961 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 7021 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 7081 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 7141 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 7201 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 7261 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 7321 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7381 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7441 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7501 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7561 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7621 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 7681 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 7741 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 7801 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 7861 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 7921 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc 7981 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa 8041 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 8101 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 8161 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 8221 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 8281 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 8341 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 8401 gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat c //