LOCUS Exported 5280 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector; encodes GFP as well as a U6-driven CFTR-targeting gRNA (SpyCas9 scaffold). ACCESSION . VERSION . KEYWORDS pAAV-RSV-GFP-U6-CFTR 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" rep_origin complement(1..456) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(138..159) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 350..369 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 583..605 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 597..614 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 598..614 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 624..643 /label=T7 /note="T7 promoter, forward primer" promoter 624..642 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 699..828 /label=AAV2 ITR primer_bind complement(901..920) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 1197..1458 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" regulatory 1496..1505 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1502..2221 /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 1502..2218 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1547..1568) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1808..1827) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2155..2176 /label=EGFP-C /note="EGFP, forward primer" promoter complement(2415..2655) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(2465..2484) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(2635..2655) /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind complement(3092..3112) /label=T3 /note="T3 promoter, forward primer" promoter complement(3092..3110) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(3131..3147) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3131..3147) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3144..3166) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3155..3171 /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)." promoter complement(3179..3209) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3224..3245 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3362..3379) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3533..4121) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3613..3632) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4292..5152) /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 4915..4934 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5153..5257) /gene="bla" /label=AmpR promoter ORIGIN 1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcacwttc aggctgcgca 481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 601 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg gagctctcta 661 gaatgcaggg gggggggggg ggggkgncca ctccctctct gcgcgctcgc tcgctcactg 721 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg 781 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctca gatctgatat 841 cgtcgaccaa ttctcatgtt tgacagctta tcatcgcaga tccgtatggt gcactctcag 901 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga 961 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa 1021 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag 1081 atatacgcgt atctgagggg actagggtgt gtttaggcga aaagcggggc ttcggttgta 1141 cgcggttagg agtcccctca ggatatagta gtttcgcttt tgcataggga gggggaaatg 1201 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc 1261 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt 1321 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattccgc 1381 attgcagaga tattgtattt aagtgcctag ctcgatacaa taaacgccat ttgaccattc 1441 accacattgg tgtgcacctc caagctgggt accgcgggcc cgggatccac cggtcgccac 1501 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga 1561 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta 1621 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac 1681 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa 1741 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt 1801 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct 1861 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca 1921 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa 1981 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc 2041 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca 2101 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt 2161 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta 2221 aaggctagca ataaaggatc gtttattttc attggaagcg tgtgttggtt ttttgatcac 2281 tcgaggcggc cgcaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact 2341 agccttattt aaacttgcta tgctgtttcc agcatagctc ttaaacggat tatgcctggc 2401 accattggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca 2461 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc 2521 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa 2581 attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat 2641 gggaaatagg ccctcttcct gcccgacctt ggcgcgcgct cggcgcgcgg tcacgctccg 2701 tcacgtggtg cgttttgcct gcgcgtcttt ccactggggc cggggcggcg cggagggttc 2761 agacgcgggc tcttactcaa acgctgatcc ccgaaactac cgagcagtgt ctgcgcgacg 2821 cgaaagcatt tttggagcga ggggccatgg tgcccatggg attcccacgg gtgttggact 2881 cgggcgcgcc gatatcagat ctgggaaacc agatgatgga ggtacccact ccctctatgc 2941 gcgctcgctc actcactcgg ccctggagac caaaggtctc cagsctggag accaaaggtc 3001 tccagggccg agtgagtgag cgagcgcgca tagagggagt gggtaggacg cgtcctgcag 3061 gatgcatact agtggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg 3121 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 3181 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 3241 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 3301 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 3361 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 3421 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 3481 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 3541 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 3601 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 3661 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 3721 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 3781 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 3841 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 3901 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 3961 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 4021 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4081 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 4141 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 4201 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 4261 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 4321 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 4381 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 4441 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 4501 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 4561 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 4621 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 4681 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 4741 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 4801 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 4861 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 4921 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 4981 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 5041 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 5101 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 5161 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 5221 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac //