LOCUS Exported 7570 bp ds-DNA circular SYN 23-NOV-2021 DEFINITION Expression of sgRNA under Ae. albopictus U6 (AALF029744) promoter. ACCESSION . VERSION . KEYWORDS pLib6.4-Aalb_744 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7570) AUTHORS Viswanatha R, Mameli E, Rodiger J, Merckaert P, Feitosa-Suntheimer F, Colpitts TM, Mohr SE, Hu Y, Perrimon N TITLE Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos JOURNAL bioRxiv 2021.03.29.437496v2 REFERENCE 2 (bases 1 to 7570) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 23, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7570 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 96..200 /gene="bla" /label=AmpR promoter CDS 201..1061 /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(419..438) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1232..1820 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1721..1740 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1974..1991 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2108..2129 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2144..2174 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2182..2198 /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 2187..2209 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2206..2222 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2206..2222 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 2276..2309 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" misc_RNA 2539..2614 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2853..5362 /label=Ac5 promoter /note="Drosophila melanogaster actin 5C promoter" CDS 5394..6110 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(5439..5460) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 6047..6068 /label=EGFP-C /note="EGFP, forward primer" CDS 6114..6167 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 6192..6791 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(6192..6211) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6688..6708 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6799..6933 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6823..6842) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 6877..6896 /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind 7127..7160 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" primer_bind complement(7176..7193) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7176..7192) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7185..7207) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(7401..7420) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7520..7542 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221 accatgatta cgccaagctt gcatgcctgc aggtcgactc tagaggatcc cccgggtgcc 2281 agggcgtgcc cttgggctcc ccgggcgcgt acttcgaaca aattcttaca gccaacacac 2341 gcgcacgcgg atgttcacta acaggtatac gtttatcgct tcatgcattc attctcgtcg 2401 agtacccgga ctgtttgcct ggacagcagt gttcggtaag tggcatatcg ccgtctattc 2461 atcgctagca ccattttgat ggctgcctgt tatatatact gggtttttcg ttctccgctc 2521 gggtcttcga gaagacctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg 2581 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttgtgaatct tcactacgtg 2641 atggctggcg tgcatatttt tttgcctacc tggagcctga gagttgttca ataaaataaa 2701 aatgtttcgt ttttttgctt tcgccagtat ttattatttt tcatcaatat gtattcaatt 2761 tggtatgtat ttagtaattg taatatatag acaaggtacc cagcatgcaa ttctatattc 2821 taaaaacaca aatgatactt ctaaaaaaaa atcatgaatg gcatcaactc tgaatcaaat 2881 ctttgcagat gcacctactt ctcatttcca ctgtcacatc atttttccag atctcgctgc 2941 ctgttatgtg gcccacaaac caagacacgt tttatggcca ttaaagctgg ctgatcgtcg 3001 ccaaacacca aatacataat gaatatgtac acattcgaga aagaagcgat caaagaagca 3061 tcttcgggcg gagtaggaga atgcggagga gaaggagaac gagctgatct agtatctctc 3121 cacaatccaa tgccaactga ccaactggcc atattcggag caatttgaag ccaatttcca 3181 tcgcctggcg atcgctccat tcttggctat atgtttttca ccgttacccg gggccatttt 3241 caaagactcg tcggcaagat aagattgtgt cactcgctgt ctctcttcat ttgtcgaaga 3301 atgctgagga atttcgcgat gacgtcggcg agtattttga agaatgagaa taatttgtat 3361 ttatacgaaa atcagttagt ggaattttct acaaaaacat gttatctata gataattttg 3421 ttgcaaaata tgttgactat gacaaagatt gtatgtatat acctttaatg tattctcatt 3481 ttcttatgta tttataatgg caatgatgat actgatgata ttttaagatg atgccagacc 3541 aaaaggcttg aatttctgcg tcttttgccg aacgcagtgc atgtgcaatt gttgtttttt 3601 ggaatattca attttcggac tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc 3661 aaagtaaagc caaaaaagcg agatggcaat accaaatgcg gcaaaacggt agtggaagga 3721 aaggggtgcg gggcagcgga aggaagggtg gggcggggcg tggcggggtc tgtggctggg 3781 cgcgacgtca ccgacgttgg agccactcct ttgaccatgt gtgcgtgtgt gtattattcg 3841 tgtctcgcca ctcgccggtt gtttttttct ttttatgctg cgctctctct agcgccatct 3901 cgcttacgca tgctcaacgc accgcatgtt gccgtttcct tttatgcgtc attttggctc 3961 gaaataggca attatttaaa caaagattag tcaacgaaaa cgctaaaata aataagtcta 4021 caatatggtt acttattgcc atgtgtgtgc agccaacgat agcaacaaaa gcaacaacac 4081 aggtggcttt ccctctttca ctttttgttt gcaagccgcg tgcgagcaag acggcacgac 4141 cggcaaacgc aattacgctg acaaagagca gacgaagttt tggcgaaaaa catcaaggcg 4201 cctgatacga atgcatttgc aataacaatt gcgatattta atattgttta tgaagctgtt 4261 tgacttcaaa acacacaaaa aaaaaaataa aacaaattat ttgaaagaga attaggaatc 4321 ggacgcttat cgttagggta acaacaagaa atgcttactg agtcacagcc tctggaaaac 4381 tgccgcaagc cagagagaga gagaaaaaga gggagagcag cttagaccgc atgtgcttgt 4441 gtgtgaggcg tctctctctt cgtctctgtt gcgcaaacgc atagactgca ctgaaaaaat 4501 cgattaccta ttttttatga atgaatattt gcactattac tattcaaaac tattaagata 4561 gcaatcacat tcaatagcca aatactatac cacctgagcg atgcaacgaa atgatcaatt 4621 tgagcaaaaa tgctgcatat ttaggacggc atcattatag aaatgcttct tgctgtgtac 4681 ttttctctcg tctggcagct gtttcgccgt tattgttaaa accggcttaa gttaggtgtg 4741 ttttctacga ctagtgaatg ccctactaga agatgtgtgt tgcacaaaat gtccctggaa 4801 taaccaattt gaagtgcaga tagcagtaaa cgtaagctaa tatgaatatt atttaactgt 4861 aatgttttaa tatcgctgga cattactaat aaacccacta taaacacatg tacatatgta 4921 tgttttggca tacaatgagt agttggggaa aaaatgtgta aaagcaccgt gaccatcaca 4981 gcataaagat aaccagctga agtatcgaat atgagtaacc cccaaattga atcacatgcc 5041 gcaactgata ggacccatgg aagtacactc ttcatggcga tatacaagac acacacaagc 5101 acgaacaccc agttgcggag gaaattctcc gtaaatgaaa acccaatcgg cgaacaattc 5161 atacccatat atggtaaaag ttttgaacgc gacttgagag cggagagcat tgcggctgat 5221 aaggttttag cgctaagcgg gctttataaa acgggctgcg ggaccagttt tcatatcact 5281 accgtttgag ttcttgtgct gtgtggatac tcctcccgac acaaagccgc tccatcagcc 5341 agcagtcgtc taatccagag accccggatc caggaattcg cccttgttta aacatggtga 5401 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 5461 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 5521 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 5581 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 5641 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 5701 gcgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 5761 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 5821 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 5881 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 5941 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 6001 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 6061 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag cttgagggca 6121 gaggaagcct tctaacatgc ggtgacgtgg aggagaatcc cggccctgct agcggtagcg 6181 gcagcggtag catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 6241 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 6301 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 6361 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga 6421 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 6481 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 6541 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca 6601 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 6661 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca 6721 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 6781 ccggtgcctg actcgaggga tccagacatg ataagataca ttgatgagtt tggacaaacc 6841 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 6901 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 6961 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 7021 ggtatggctg attatgatca gtcgaccgat gcccttgaga gccttcaacc cagtcagctc 7081 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atggtacgcg cccggggagc 7141 ccaagggcac gccctggcac ccgaagggcg aattcactgg ccgtcgtttt acaacgtcgt 7201 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 7261 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 7321 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 7381 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 7441 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 7501 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 7561 aaacgcgcga //