LOCUS Exported 7800 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dSa Cas9 fused to optimized combination of truncated activation domains with syn pA (poly adenylation) signal. ACCESSION . VERSION . KEYWORDS pAAV-CMV-dSa-VPR mini.-syn pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7800) TITLE Church Lab CRISPR plasmids 2017 REFERENCE 2 (bases 1 to 7800) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7800 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 19..38 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(55..510) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(192..213) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 404..423 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 585..725 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 596..725 /label=AAV2 ITR enhancer 745..1124 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1125..1328 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1279..1299 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 1347..1356 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1359..1379 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4560..4607 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4638..4787 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 4812..4832 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4992..5348 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" polyA_signal 5586..5634 /note="synthetic polyadenylation signal" repeat_region 5647..5787 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5647..5776 /label=AAV2 ITR rep_origin complement(5849..6437) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5929..5948) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6608..7468) /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 7231..7250 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7469..7573) /gene="bla" /label=AmpR promoter primer_bind 7641..7659 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7697..7719) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg 61 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 121 accaatagac cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagagt 181 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 241 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa 301 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 361 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 421 gagcgggcgc taaggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 481 ccgcgcttaa tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga 541 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg 601 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg 661 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg 721 ttcctgcggc cgcctcgagg cgttgacatt gattattgac tagttattaa tagtaatcaa 781 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 841 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 901 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 961 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 1021 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 1081 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 1141 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 1201 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 1261 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 1321 gcagagctct ctggctaact accggtgcca ccatggcccc aaagaagaag cggaaggtcg 1381 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggcc atcggcatca 1441 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc 1501 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca 1561 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact 1621 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga 1681 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca 1741 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca 1801 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc 1861 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca 1921 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg 1981 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg 2041 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga 2101 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg 2161 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga 2221 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc 2281 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca 2341 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg 2401 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga 2461 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg 2521 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca 2581 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc 2641 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga 2701 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct 2761 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca 2821 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga 2881 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca 2941 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt 3001 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg 3061 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc 3121 tcgtgaagca ggaagaagcc agcaagaagg gcaaccggac cccattccag tacctgagca 3181 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg 3241 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca 3301 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca 3361 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga 3421 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc 3481 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca 3541 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg 3601 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct 3661 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc 3721 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg 3781 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg 3841 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc 3901 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc 3961 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg 4021 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca 4081 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat 4141 tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga 4201 tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga 4261 agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga 4321 tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag 4381 tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc 4441 ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca 4501 ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggca 4561 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tccgaggcca 4621 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg 4681 ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg 4741 acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa 4801 gttccggatc tccgaaaaag aaacgcaaag ttggttcggg aggtggttcg ggtggctctg 4861 gatcagtgct gcctcaggct cctgctcctg caccagctcc agccatggtg tctgcactgg 4921 ctcaggcacc agcacccgtg cctgtgctgg ctcctggacc tccacaggct gtggctccac 4981 cagcccctaa acctacacag gccggcgagg gcacactgtc tgaagctctg ctgcagctgc 5041 agttcgacga cgaggatctg ggagccctgc tgggaaacag caccgatcct gccgtgttca 5101 ccgacctggc cagcgtggac aacagcgagt tccagcagct gctgaaccag ggcatccctg 5161 tggcccctca caccaccgag cccatgctga tggaataccc cgaggccatc acccggctcg 5221 tgacaggcgc tcagaggcct cctgatccag ctcctgcccc tctgggagca ccaggcctgc 5281 ctaatggact gctgtctggc gacgaggact tcagctctat cgccgatatg gatttctcag 5341 ccttgctgtc aggcggtggt agtggtggga gcggtagtga cctttcccat ccgcccccaa 5401 ggggccatct ggatgagctg acaaccacac ttgagtccat gaccgaggat ctgaacctgg 5461 actcacccct gaccccggaa ttgaacgaga ttctggatac cttcctgaac gacgagtgcc 5521 tcttgcatgc catgcatatc agcacaggac tgtccatctt cgacacatct ctgttttagg 5581 aattcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgcaattg 5641 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 5701 ctgaggccgg gcgaccaaag gttgcccgac gcccgggctt tgcccgggcg gcctcagtga 5761 gcgagcgagc gcgcagctgc ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga 5821 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 5881 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 5941 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 6001 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 6061 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 6121 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 6181 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 6241 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 6301 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 6361 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 6421 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 6481 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 6541 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 6601 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 6661 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 6721 ctggccccag tgctgcaatg ataccgcggc ttccacgctc accggctcca gatttatcag 6781 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 6841 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 6901 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 6961 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7021 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7081 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7141 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7201 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7261 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 7321 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 7381 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 7441 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 7501 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 7561 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 7621 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg 7681 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 7741 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc //