LOCUS Exported 5168 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION AAV vector for expression of AcrIIA4 (no miR binding sites, control vector). ACCESSION . VERSION . KEYWORDS AAV CMV-driven AcrIIA4-scaffold (2xBsmBI sites) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5168) 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 5168) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5168 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" enhancer 397..776 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 777..980 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 931..951 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" promoter 1025..1043 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1065..1088 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" primer_bind complement(1481..1498) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1487..1711 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter complement(1922..1940) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1950..1967) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1950..1966) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1959..1981) /label=M13/pUC Forward /note="In lacZ gene" rep_origin complement(2107..2562) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2244..2265) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2456..2475 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2589..2693 /gene="bla" /label=AmpR promoter CDS 2694..3554 /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 complement(2912..2931) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3725..4313 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4214..4233 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4467..4484 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4601..4622 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4637..4667 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4675..4691 /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)." primer_bind 4680..4702 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4699..4715 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4699..4715 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4734..4754 /label=T3 /note="T3 promoter, forward primer" promoter 4736..4754 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg agtcttcgag gaagacaggt ttaagagcta tgctggaaac agcatagcaa 301 gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 361 tgcggccgcg gccgctctac aaatgtggta tctagagaca ttgattattg actagttatt 421 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 481 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 541 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 601 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 661 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 721 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 781 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 841 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 901 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 961 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg 1021 aaattaatac gactcactat agggagaccc aagctggcta gatggactat aaggacgatg 1081 acgataaagc tggcggcagc atgaacatta acgacctgat tcgggaaata aagaacaagg 1141 actacacggt caagctgtca ggcaccgact ccaactctat aacacagctc attataaggg 1201 ttaataatga tggcaacgaa tatgtcatct ctgaatccga gaatgaatca atcgtggaga 1261 aattcatcag tgcatttaaa aacggctgga accaggagta cgaagatgag gaagagttct 1321 acaatgatat gcaaactatc acactgaaat ctgagctgaa ctagctcgag cggccgccac 1381 tgtgctggat atctgcagaa ttagagacgc cgttgatgtt gaagtttata taccgttgat 1441 gttgaagtcg tctctagctt aagtttaaac cgctgatcag cctcgactgt gccttctagt 1501 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 1561 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 1621 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 1681 aggcatgctg gggatgcggt gggctctatg ggtcgactcg acgatatcag atctgaggaa 1741 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc 1801 cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg 1861 cgcagagagg gagtggccnc cccccccccc cccccctgca ttctagagag ctccaattcg 1921 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg 1981 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 2041 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 2101 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 2161 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 2221 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 2281 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 2341 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 2401 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 2461 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 2521 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg 2581 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 2641 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 2701 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 2761 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 2821 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 2881 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 2941 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3001 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3061 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 3181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 3241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 3301 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 3361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 3421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 3481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 3541 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 3601 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 3661 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 3721 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 3781 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 3841 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 3901 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 3961 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4021 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4081 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 4141 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4201 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 4261 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 4321 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 4381 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 4441 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 4501 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 4561 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 4621 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 4681 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta 4741 accctcacta aagggaacaa aagctgggta ccactagtat gcatcctgca ggacgcgtcc 4801 tacccactcc ctctatgcgc gctcgctcac tcactcggcc ctggagacca aaggtctcca 4861 gggccgagtg agtgagcgag cgcgcataga gggagtgggt acctccatca tctggtttcc 4921 cagatctgat atcggcgcgc ccgagtccaa cacccgtggg aatcccatgg gcaccatggc 4981 ccctcgctcc aaaaatgctt tcgcgtcgcg cagacactgc tcggtagttt cggggatcag 5041 cgtttgagta agagcccgcg tctgaaccct ccgcgccgcc ccggccccag tggaaagacg 5101 cgcaggcaaa acgcaccacg tgacggagcg tgaccgcgcg ccgagcgcgc gccaaggtcg 5161 ggcaggaa //