LOCUS Exported 5281 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 5281) 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 5281) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5281 /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(3061..3780) /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(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)" promoter complement(3824..4085) /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" primer_bind 4362..4381 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(4639..4658) /label=T7 /note="T7 promoter, forward primer" promoter complement(4640..4658) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4668..4685) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4668..4684) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4677..4699) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4826..5281 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4913..4932) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5123..5144 /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 gaccaaaggt ctccgccctg gagaccaaag 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 caccgtgaca 2881 tcaattatta tacatgttta 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 ggcagcagca 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 gttaccatgt tgcaagacta caagagtatt gcataagact 4081 acatttcccc ctccctatgc aaaagcgaaa ctactatatc ctgaggggac tcctaaccgc 4141 gtacaaccga agccccgctt ttcgcctaaa cacaccctag tcccctcaga tacgcgtata 4201 tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca 4261 attgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac 4321 ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt 4381 actgagagtg caccatacgg atctgcgatg ataagctgtc aaacatgaga attggtcgac 4441 gatatcagat ctgaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 4501 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 4561 tcagtgagcg agcgagcgcg carvrrggrg ggggggmsyc ccccccyccc ccyctgcatt 4621 ctagagagct ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt 4681 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 4741 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 4801 ttgcgcagcc tgaatgggcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt 4861 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4921 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4981 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 5041 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 5101 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 5161 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 5221 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 5281 t //