LOCUS Exported 5284 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector; encodes GFP as well as a U6-driven EMX1-targeting gRNA (SpyCas9 scaffold). ACCESSION . VERSION . KEYWORDS pAAV-RSV-GFP-U6-EMX1 gRNA (SpyCas9 scaffold) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5284) AUTHORS Bubeck F, Hoffmann MD, Harteveld Z, Aschenbrenner S, Bietz A, Waldhauer MC, Borner K, Fakhiri J, Schmelas C, Dietz L, Grimm D, Correia BE, Eils R, Niopek D TITLE Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. JOURNAL Nat Methods. 2018 Oct 30. pii: 10.1038/s41592-018-0178-9. doi: 10.1038/s41592-018-0178-9. PUBMED 30377362 REFERENCE 2 (bases 1 to 5284) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5284 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 24..128 /gene="bla" /label=AmpR promoter CDS 129..989 /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(347..366) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1160..1748 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1649..1668 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1902..1919 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2036..2057 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2072..2102 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2110..2126 /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 2115..2137 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2134..2150 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2134..2150 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2169..2189 /label=T3 /note="T3 promoter, forward primer" promoter 2171..2189 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 2627..2867 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2627..2647 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2798..2817 /label=LKO.1 5' /note="Human U6 promoter, forward primer" CDS complement(3064..3780) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3106..3127) /label=EGFP-C /note="EGFP, forward primer" primer_bind 3455..3474 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3714..3735 /label=EGFP-N /note="EGFP, reverse primer" regulatory 3777..3786 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 4364..4383 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(4640..4659) /label=T7 /note="T7 promoter, forward primer" promoter complement(4641..4659) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4669..4686) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4669..4685) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4678..4700) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4827..5282 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4914..4933) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5124..5145 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccact agtatgcatc 2221 ctgcaggacg cgtcctaccc actccctcta tgcgcgctcg ctcactcact cggccctgga 2281 gaccaaaggt ctccagactg ccggcctctg gccggcaggg ccgagtgagt gagcgagcgc 2341 gcatagaggg agtgggtacc tccatcatct aggtttgcca gatctgatat cggcgcgccc 2401 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc 2461 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc 2521 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg 2581 acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg gcctatttcc 2641 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa 2701 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct 2761 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac 2821 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgagtc 2881 cgagcagaag aagaagttta agagctatgc tggaaacagc atagcaagtt taaataaggc 2941 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttgc ggccgcctcg 3001 agtgatcaaa aaaccaacac acgcttccaa tgaaaataaa cgatccttta ttgctagcct 3061 ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag 3121 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta 3181 gtggttgtcg ggcagcaaca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc 3241 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc 3301 gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt 3361 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac 3421 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa 3481 gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg 3541 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag 3601 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc 3661 gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc 3721 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat 3781 ggtggcgacc ggtggatccc gggcccgcgg tacccagctt ggaggtgcac accaatgtgg 3841 tgaatggtca aatggcgttt attgtatcga gctaggcact taaatacaat atctctgcaa 3901 tgcggaattc agtggttcgt ccaatccatg tcagacccgt ctgttgcctt cctaataagg 3961 cacgatcgta ccaccttact tccaccaatc ggcatgcacg gtgctttttc tctccttgta 4021 aggcatgttg ctaactcatc cttaccatgt tgcaagacta ccaagagtat ttgcataaga 4081 ctacatttcc ccctccctat gcaaaagcga aactactata tcctgagggg actcctaacc 4141 gcgtacaacc gaagccccgc ttttcgccta aacacaccct agtcccctca gatacgcgta 4201 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg 4261 caattgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg 4321 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt 4381 gtactgagag tgcaccatac ggatctgcga tgataagctg tcaaacatga gaattggtcg 4441 acgatatcag atctaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 4501 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc 4561 ctcagtgagc gagcgagcgc gcagagaggg agtggccccc cccccccccc ccccctgcat 4621 tctagagagc tccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 4681 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4741 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4801 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg 4861 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4921 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4981 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5041 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5101 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5161 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 5221 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat 5281 ttag //