LOCUS Exported 5280 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 5280) 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 5280) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5280 /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 2626..2866 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2626..2646 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2797..2816 /label=LKO.1 5' /note="Human U6 promoter, forward primer" CDS complement(3060..3779) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS complement(3063..3779) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3105..3126) /label=EGFP-C /note="EGFP, forward primer" primer_bind 3454..3473 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3713..3734 /label=EGFP-N /note="EGFP, reverse primer" regulatory 3776..3785 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(3823..4084) /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" primer_bind 4361..4380 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" repeat_region 4453..4582 /label=AAV2 ITR primer_bind complement(4638..4657) /label=T7 /note="T7 promoter, forward primer" promoter complement(4639..4657) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4667..4684) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4667..4683) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4676..4698) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4825..5280 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4912..4931) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5122..5143 /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 tactgttctt 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 gacctttggt ctccgccctg gagacctttg gtctccaggg ccgagtgagt gagcgagcgc 2341 gcatagaggg agtgggtacc tccatcatct ggtttcccag atctgatatc ggcgcgcccg 2401 agtccaacac ccgtgggaat cccatgggca ccatggcccc tcgctccaaa aatgctttcg 2461 cgtcgcgcag acactgctcg gtagtttcgg ggatcagcgt ttgagtaaga gcccgcgtct 2521 gaaccctccg cgccgccccg gccccagtgg aaagacgcgc aggcaaaacg caccacgtga 2581 cggagcgtga ccgcgcgccg agcgcgcgcc aaggtcgggc aggaagaggg cctatttccc 2641 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat 2701 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 2761 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 2821 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgagtcc 2881 gagcagaaga agaagtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct 2941 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgcg gccgcctcga 3001 gtgatcaaaa aaccaacaca cgcttccaat gaaaataaac gatcctttat tgctagcctt 3061 tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg 3121 accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag 3181 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg 3241 gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg 3301 ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg 3361 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc 3421 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag 3481 aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc 3541 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg 3601 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg 3661 taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg 3721 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg 3781 gtggcgaccg gtggatcccg ggcccgcggt acccagcttg gaggtgcaca ccaatgtggt 3841 gaatggtcaa atggcgttta ttgtatcgag ctaggcactt aaatacaata tctctgcaat 3901 gcggaattca gtggttcgtc caatccatgt cagacccgtc tgttgccttc ctaataaggc 3961 acgatcgtac caccttactt ccaccaatcg gcatgcacgg tgctttttct ctccttgtaa 4021 ggcatgttgc taactcatcg ttaccatgtt gcaagactac aagagtattg cataagacta 4081 catttccccc tccctatgca aaagcgaaac tactatatcc tgaggggact cctaaccgcg 4141 tacaaccgaa gccccgcttt tcgcctaaac acaccctagt cccctcagat acgcgtatat 4201 ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa 4261 ttgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc 4321 tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta 4381 ctgagagtgc accatacgga tctgcgatga taagctgtca aacatgagaa ttggtcgacg 4441 atatcagatc tgaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 4501 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 4561 cagtgagcga gcgagcgcgc agagagggag tggccccccc cccccccccc ccntgcattc 4621 tagagagctc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 4681 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 4741 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 4801 tgcgcagcct gaatggsgca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg 4861 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 4921 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 4981 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 5041 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 5101 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 5161 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 5221 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt //