LOCUS Exported 7591 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION AAV Vector for expression of N-terminal SpyCas9 fragment with Intein and a U6-driven F+E gRNA scaffold. ACCESSION . VERSION . KEYWORDS AAV N-SpyCas9-Intein, U6-gRNA scaffold (F+E) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7591) AUTHORS Hoffmann MD, Aschenbrenner S, Grosse S, Rapti K, Domenger C, Fakhiri J, Mastel M, Borner K, Eils R, Grimm D, Niopek D TITLE Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins. JOURNAL Nucleic Acids Res. 2019 Apr 15. pii: 5458120. doi: 10.1093/nar/gkz271. PUBMED 30982889 REFERENCE 2 (bases 1 to 7591) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7591 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(7..27) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" enhancer 257..636 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 637..840 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 791..811 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" intron 932..1064 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" regulatory 1094..1103 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1106..1126 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1151..2866 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" primer_bind complement(3606..3625) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" polyA_signal 3635..3716 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind 3660..3679 /label=SV40pA-R /note="SV40 polyA, reverse primer" promoter 4065..4305 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4065..4085 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4236..4255 /label=LKO.1 5' /note="Human U6 promoter, forward primer" repeat_region 4480..4609 /label=AAV2 ITR primer_bind complement(4636..4652) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4636..4652) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4649..4671) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4660..4676 /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(4684..4714) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4729..4750 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4867..4884) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5038..5626) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5118..5137) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5797..6657) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6420..6439 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6658..6762) /gene="bla" /label=AmpR promoter rep_origin complement(6789..7244) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6926..6947) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7138..7157 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7371..7393 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7385..7402 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7386..7402 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 7421..7550 /label=AAV2 ITR ORIGIN 1 ggccgcggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 61 gtatgcaaag catgcatctc aattagtcag caaccagtcc cggtctcctc ccatgcatgt 121 caatattggc cattagccat attattcatt ggttatatag cataaatcaa tattggctat 181 tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca 241 atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc aattacgggg 301 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 361 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 421 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 481 cacttggcag tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac 541 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg 601 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc 661 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 721 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc 781 gccccgttga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagaggt 841 cgtttagtga accgtcagat cactagtagc tttattgcgg tagtttatca cagttaaatt 901 gctaacgcag tcagtgctcg actgatcaca ggtaagtatc aaggttacaa gacaggttta 961 aggaggccaa tagaaactgg gcttgtcgag acagagaaga ttcttgcgtt tctgataggc 1021 acctattggt cttactgaca tccactttgc ctttctctcc acaggggtac cgaagccgct 1081 agcgctaccg gtcgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt 1141 cccagcagcc gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg 1201 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac 1261 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggaaac 1321 ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg 1381 gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt 1441 ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat 1501 ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct 1561 gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct 1621 ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa 1681 cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga 1741 gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa 1801 atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt 1861 tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct 1921 ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct 1981 gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga 2041 cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag 2101 cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct 2161 tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg 2221 ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc 2281 catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct 2341 gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga 2401 actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga 2461 aaagattgag aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg 2521 aaattccaga ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt 2581 cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt 2641 tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt 2701 cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc 2761 attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa 2821 agttaccgtg aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt 2881 tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct 2941 gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga 3001 cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac 3061 ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg 3121 atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac 3181 aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca 3241 tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttgtc tggctggcga 3301 tactctcatt accctggccg atggacgacg agtgcctatt agagaactgg tgtcacagca 3361 gaatttttcc gtgtgggctc tgaatcctca gacttaccgc ctggagaggg ctagagtgag 3421 tagagctttc tgtaccggca tcaaacctgt gtaccgcctc accactagac tggggagatc 3481 cattagggcc actgccaacc accgatttct cacacctcag ggctggaaac gagtcgatga 3541 actccagcct ggagattacc tggctctgcc taggagaatc cctactgcct cctgatctcg 3601 agctcgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg 3661 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca 3721 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa 3781 gcaagtaaaa cctctacaaa tgtggtactt aagaggggga gaccaaaggg cacgcgtcga 3841 gtccaacacc cgtgggaatc ccatgggcac catggcccct cgctccaaaa atgctttcgc 3901 gtcgcgcaga cactgctcgg tagtttcggg gatcagcgtt tgagtaagag cccgcgtctg 3961 aaccctccgc gccgccccgg ccccagtgga aagacgcgca ggcaaaacgc accacgtgac 4021 ggagcgtgac cgcgcgccga gcgcgcgcca aggtcgggca ggaagagggc ctatttccca 4081 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt 4141 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg 4201 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt 4261 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgagtctt 4321 cgaggaagac aggtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag 4381 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttctaga gggcggccgc 4441 aggtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt 4501 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc 4561 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc cttaattaaa 4621 tctggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 4681 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 4741 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 4801 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 4861 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 4921 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4981 gtgagcaaaa ccgcagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 5041 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 5101 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 5161 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 5221 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5281 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5341 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5401 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5461 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 5521 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 5581 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5641 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 5701 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 5761 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 5821 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5881 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 5941 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 6001 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 6061 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttaccatta ctacaggcat 6121 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 6181 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 6241 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 6301 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 6361 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 6421 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 6481 gcgaaaactc tcaaggatct taccactatt gagatccagt tcgatgtaac ccactcgtgc 6541 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6601 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 6661 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 6721 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaagat 6781 gccacctgaa attataaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 6841 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata 6901 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt 6961 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc 7021 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa 7081 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg 7141 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt 7201 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag 7261 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 7321 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 7381 acgttgtaaa acgacggcca gtgaattagg ttaattaagg ctgcgcgctc gctcgctcac 7441 tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag 7501 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct tgtagttaat 7561 gattaacccg ccatgctact tatctacctg c //