LOCUS Exported 8451 bp ds-DNA circular SYN 19-JAN-2022 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 Jan 19, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8451 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 165..544 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 545..748 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 699..719 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 790..809 /label=T7 /note="T7 promoter, forward primer" promoter 790..808 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 816..825 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 822..2540 /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 4980..5000 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5013..5078 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 5097..5153 /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 5154..5870 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(5199..5220) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5460..5479) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5807..5828 /label=EGFP-C /note="EGFP, forward primer" CDS 5879..5896 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5919..5936) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5925..6149 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6220..6236) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6220..6236) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6233..6255) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6244..6260 /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(6268..6298) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6313..6334 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6451..6468) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6622..7210) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6702..6721) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7381..8241) /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 8004..8023 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8242..8346) /gene="bla" /label=AmpR promoter primer_bind complement(8425..8444) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat 61 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag 121 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac 181 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 241 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 301 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 361 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 421 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 481 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 541 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 601 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 661 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 721 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc 781 cgcggccgct aatacgactc actataggga gagccgccac catggacaag aagtacagca 841 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg 901 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc 961 tgatcggagc cctgctgttc gacagcggcg aaacagccga gagaacccgg ctgaagagaa 1021 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca 1081 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg 1141 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg 1201 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg 1261 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc 1321 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc 1381 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg 1441 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg 1501 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg 1561 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga 1621 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg 1681 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga 1741 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg 1801 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt 1861 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag 1921 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg 1981 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca 2041 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg 2101 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc 2161 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca 2221 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt 2281 ccgcccagag cttcatcgag cggatgaccg ccttcgataa gaacctgccc aacgagaagg 2341 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag 2401 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg 2461 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg 2521 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt 2581 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc 2641 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg 2701 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag 2761 tgatgaagca gctgaagcgg cggagataca ccggctgggg cgccctgagc cggaagctga 2821 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg 2881 gcttcgccaa cagaaacttc atggccctga tccacgacga cagcctgacc tttaaagagg 2941 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc 3001 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc 3061 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga 3121 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg 3181 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc 3241 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg 3301 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc 3361 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga 3421 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc 3481 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg 3541 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccgggcca 3601 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg 3661 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc 3721 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg 3781 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg 3841 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc 3901 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca 3961 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg 4021 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc 4081 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca 4141 aagagtctat cagacccaag aggaacagcg ataagctgat cgccagaaag aaggactggg 4201 accctaagaa gtacggcggc ttcctgtggc ccaccgtggc ctattctgtg ctggtggtgg 4261 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca 4321 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct 4381 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg 4441 aaaacggccg gaagagaatg ctggcctctg ccaagcagct gcagaaggga aacgaactgg 4501 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg 4561 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg 4621 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc 4681 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg 4741 agaatatcat ccacctgttt accctgacca gactgggagc ccctagagcc ttcaagtact 4801 ttgacaccac catcgacccc aagcagtaca gaagcaccaa agaggtgctg gacgccaccc 4861 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag 4921 gcgacggatc cggcggaggc ggaagcggga aaagaaccgc cgacggcagc gaattcgagc 4981 ccaagaagaa gaggaaagtc tcgagcggag gcgactacaa agaccatgac ggtgattata 5041 aagatcatga catcgattac aaggatgacg atgacaaggg aggcggtgga agcggcgcaa 5101 caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgatggtga 5161 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 5221 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 5281 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 5341 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 5401 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 5461 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 5521 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 5581 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 5641 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 5701 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 5761 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 5821 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tgaccggtca 5881 tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 5941 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 6001 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 6061 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 6121 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 6181 gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 6241 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 6301 ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 6361 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 6421 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 6481 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 6541 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 6601 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 6661 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 6721 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 6781 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 6841 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 6901 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 6961 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 7021 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 7081 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 7141 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 7201 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 7261 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 7321 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 7381 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 7441 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 7501 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 7561 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 7621 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 7681 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 7741 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 7801 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 7861 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 7921 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 7981 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 8041 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 8101 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 8161 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 8221 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 8281 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 8341 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc 8401 cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt t //