LOCUS Exported 5285 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector; encodes GFP as well as a U6-driven CCR5-targeting gRNA (SpyCas9 scaffold). ACCESSION . VERSION . KEYWORDS pAAV-RSV-GFP-U6-CCR5 gRNA (SpyCas9 scaffold) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5285) 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 5285) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5285 /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(3065..3781) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3107..3128) /label=EGFP-C /note="EGFP, forward primer" primer_bind 3456..3475 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3715..3736 /label=EGFP-N /note="EGFP, reverse primer" regulatory 3778..3787 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 4365..4384 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(4641..4660) /label=T7 /note="T7 promoter, forward primer" promoter complement(4642..4660) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4670..4687) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4670..4686) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4679..4701) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4828..5283 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4915..4934) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5125..5146 /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 acaccgtgac 2881 atcaattatt atacatgttt aagagctatg ctggaaacag catagcaagt ttaaataagg 2941 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg cggccgcctc 3001 gagtgatcaa aaaaccaaca cacgcttcca atgaaaataa acgatccttt attgctagcc 3061 tttacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca 3121 ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt 3181 agtggttgtc gggcagcaac acggggccgt cgccgatggg ggtgttctgc tggtagtggt 3241 cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc 3301 cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct 3361 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca 3421 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga 3481 agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct 3541 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga 3601 gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc 3661 cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc 3721 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca 3781 tggtggcgac cggtggatcc cgggcccgcg gtacccagct tggaggtgca caccaatgtg 3841 gtgaatggtc aaatggcgtt tattgtatcg agctaggcac ttaaatacaa tatctctgca 3901 atgcggaatt cagtggttcg tccaatccat gtcagacccg tctgttgcct tcctaataag 3961 gcacgatcgt accaccttac ttccaccaat cggcatgcac ggtgcttttt ctctccttgt 4021 aaggcatgtt gctaactcat ccttaccatg ttgcaagact accaagagta tttgcataag 4081 actacatttc cccctcccta tgcaaaagcg aaactactat atcctgaggg gactcctaac 4141 cgcgtacaac cgaagccccg cttttcgcct aaacacaccc tagtcccctc agatacgcgt 4201 atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat 4261 gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc 4321 gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat 4381 tgtactgaga gtgcaccata cggatctgcg atgataagct gtcaaacatg agaattggtc 4441 gacgatatca gatctaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 4501 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg 4561 cctcagtgag cgagcgagcg cgcagagagg gagtggcccc cccccccccc cccccctgca 4621 ttctagagag ctccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg 4681 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 4741 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 4801 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 4861 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 4921 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4981 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5041 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5101 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5161 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 5221 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa 5281 tttag //