LOCUS Exported 8045 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Human expression vector for SaCas9-HF variant.. ACCESSION . VERSION . KEYWORDS pCAG-CFP-SaCas9-HF (without sgRNA) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8045) AUTHORS Tan Y, Chu AHY, Bao S, Hoang DA, Kebede FT, Xiong W, Ji M, Shi J, Zheng Z TITLE Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. JOURNAL Proc Natl Acad Sci U S A. 2019 Sep 30. pii: 1906843116. doi: 10.1073/pnas.1906843116. PUBMED 31570596 REFERENCE 2 (bases 1 to 8045) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8045 /organism="synthetic DNA construct" /mol_type="other DNA" intron 154..1170 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1094..1116 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1178..1197 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 1263..1272 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4437..4457 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4464..4529 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(4619..4638) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 4684..4739 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(4738..4757) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(5100..5116) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5100..5116) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5113..5135) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5124..5140 /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(5148..5178) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5193..5214 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5273..5468 /label=SV40 promoter /note="SV40 early promoter" rep_origin 5319..5454 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5381..5400 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 5474..5608 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5511..5530) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5565..5584 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5676..5693) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5847..6435) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5927..5946) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6606..7466) /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 7229..7248 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7467..7571) /gene="bla" /label=AmpR promoter enhancer 7602..7981 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg 61 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg 121 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc 181 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 241 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa 301 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct 361 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg 421 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg 481 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc 541 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg 601 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc 661 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg 721 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg 781 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc 841 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt 901 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg 961 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt 1021 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga 1081 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac 1141 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg 1201 tctcatcatt ttggcaaaga attctgcagt cgacggtacc gcgggcccgg gatccaccgg 1261 tcgccaccat ggggaaacgg aactacatcc tggggcttga cattgggata accagcgttg 1321 gctacggaat tattgattat gagacacgcg atgtgattga cgccggggtt aggctgttca 1381 aagaggccaa cgttgaaaac aacgagggaa gacggagtaa gcgcggagca agaagactca 1441 agcgcagacg gagacatcgg attcagaggg tgaaaaagct gctcttcgat tacaatctcc 1501 tgaccgatca tagtgagctg agcggaatca acccctacga ggcgcgagtg aaagggcttt 1561 cccagaagct gtccgaagag gagttctccg ccgcgttgct gcacctggcc aaacggaggg 1621 gggttcacaa tgtaaacgaa gtggaggagg acacgggcaa tgaacttagt acgaaagaac 1681 agatcagtag gaactctaag gctctcgaag agaaatacgt cgctgagttg cagcttgaga 1741 gactgaaaaa agacggcgaa gtacgcggat ctattaatag gttcaagact tcagattacg 1801 taaaggaagc caagcagctc ctgaaagtac agaaagcgta ccatcagctc gatcagagct 1861 tcatcgatac ctacatagat ttgctggaga cacggaggac atactacgag ggcccagggg 1921 aaggatctcc ttttgggtgg aaggacatca aggaatggta cgagatgctt atgggacatt 1981 gtacatattt tccggaggag ctcgcgagcg tcaagtacgc ctacaatgcc gacctgtaca 2041 atgccctcaa tgacctcaat aacctcgtga ttaccaggga cgagaacgag aagctggagt 2101 actatgaaaa gttccagatt atcgagaatg tgtttaagca gaagaagaag ccgacactta 2161 agcagattgc aaaggaaatc ctcgtgaatg aggaagatat caagggatac agagtgacaa 2221 gtacaggcaa gcccgagttc acaaatctga aggtgtacca cgatattaag gacataaccg 2281 cacgaaagga gataatcgaa aacgctgagc tcctcgatca gatcgcaaaa attcttacca 2341 tctaccagtc tagtgaggac attcaggagg aactgactaa tctgaacagt gagctcaccc 2401 aagaggaaat tgagcagatt tcaaacctga aaggctacac cgggacgcac aatctgagcc 2461 tcaaagcaat caacctcatt ctggatgaac tttggcacac aaatgacgcc caaattgcca 2521 tattcgcccg cctgaaactg gtgccaaaaa aagtggatct gtcacagcaa aaggaaatcc 2581 ctacaacctt ggttgacgat tttattctgt cccccgttgt caagcggagc ttcatccagt 2641 caatcaaggt gatcaatgcc atcattaaaa aatacggatt gccaaacgat ataattatcg 2701 agcttgcacg agagaagaac tcaaaggacg cccagaagat gattaacgaa atgcagaagc 2761 gcaaccgcca gacaaacgaa cgcatagagg aaattataag aacaaccggc aaagagaatg 2821 ccaagtatct gatcgagaaa atcaagctgc acgacatgca agaaggcaag tgcctgtact 2881 ctctggaagc tatcccactc gaagatctgc tgaataatcc attcaattac gaggtggacc 2941 acatcatccc tagatccgta agctttgaca attccttcaa taacaaagtt ctggttaaac 3001 aggaggaaaa ttctaaaaaa gggaaccgga ccccgttcca gtacctgagc tccagtgaca 3061 gcaagattag ctacgagact tttaagaaac atattctgaa tctggccaaa ggcaaaggca 3121 ggatcagcaa gaccaagaag gagtacctcc tcgaagaacg cgacattaac agatttagtg 3181 tgcagaaaga tttcatcaac cgaaaccttg tcgatactcg gtacgccacg gcaggcctga 3241 tgaatctcct caggagctac ttccgcgtca ataatctgga cgttaaagtc aagagcataa 3301 atgggggatt caccagcttt ctgaggagaa agtggaagtt taagaaggaa cgaaacaaag 3361 gatacaagca ccatgctgag gatgctttga tcatcgctaa cgcggacttt atctttaagg 3421 aatggaaaaa gctggataag gcaaagaaag tgatggaaaa ccagatgttc gaggagaagc 3481 aggcagagtc aatgcctgag atcgagacag agcaggaata caaggaaatt ttcatcaccc 3541 ctcatcagat taaacacata aaggacttca aagactataa atactctcat agggtggaca 3601 aaaaacccaa tcgcgagctc attaatgaca ccctgtactc aacacggaag gatgataaag 3661 gtaatacctt gattgtgaat aatcttaatg gattgtatga caaagataac gacaagctca 3721 agaagctgat caacaagtct ccagagaagc tccttatgta tcaccacgac ccacagactt 3781 atcagaaatt gaaactgatc atggagcaat acggggatga gaagaaccca ctctacaaat 3841 attatgagga aacaggtaat tacctgacca agtactccaa gaaggataac ggaccagtga 3901 tcaaaaagat aaagtactat ggcaacaaac ttaatgcgca tttggacata actgacgatt 3961 accccaattc tcgaaacaag gttgtgaagc tctccctgaa gccttataga tttgacgtgt 4021 acctggataa tggggtttat aaattcgtca ccgtgaaaaa tctggacgtg atcaaaaagg 4081 agaactatta tgaagtaaac tcaaagtgct atgaggaggc gaagaagctg aagaagatct 4141 ccaatcaggc cgagttcatc gcttccttct ataataacga tctcatcaag atcaatggag 4201 agctttatcg cgtcattggt gtgaacaatg acttgctgaa caggatcgaa gtcaatatga 4261 tagacattac ctaccgggag tatctcgaaa acatgaatga taaacggccg cctcgcatca 4321 tcaagacaat cgcatctaaa actcagtcaa taaaaaagta ctctaccgat atcctgggga 4381 atctctatga agtgaagtca aagaagcacc cacaaatcat taaaaaaggt ggatccccca 4441 agaagaagag gaaagtctcg agcgactaca aagaccatga cggtgattat aaagatcatg 4501 acatcgatta caaggatgac gatgacaagt aaagcggccg cactcctcag gtgcaggctg 4561 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc 4621 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 4681 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 4741 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 4801 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc 4861 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt 4921 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat 4981 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag 5041 ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc gtaatcatgg 5101 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 5161 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 5221 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca 5281 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 5341 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 5401 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 5461 tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 5521 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 5581 catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg 5641 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 5701 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5761 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 5821 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 5881 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 5941 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 6001 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 6061 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 6121 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 6181 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 6241 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 6301 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6361 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 6421 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 6481 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 6541 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 6601 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 6661 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 6721 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 6781 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 6841 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 6901 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 6961 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 7021 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 7081 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 7141 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 7201 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 7261 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 7321 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 7381 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 7441 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 7501 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 7561 ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt cgacattgat tattgactag 7621 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 7681 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 7741 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 7801 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 7861 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 7921 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 7981 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc 8041 aattt //