LOCUS Exported 7562 bp ds-DNA circular SYN 23-NOV-2021 DEFINITION Expression of sgRNA under An. gambiae U6-2 (AGAP013695) promoter. ACCESSION . VERSION . KEYWORDS pLib6.4B-Agam_695 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7562) 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 7562) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 23, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7562 /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 2481..2556 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2845..5354 /label=Ac5 promoter /note="Drosophila melanogaster actin 5C promoter" CDS 5386..6102 /codon_start=1 /product="enhanced blue variant of GFP (Ai et al., 2007)" /label=EBFP2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL EFRTAAGITLGMDELYK" primer_bind complement(5431..5452) /label=EGFP-N /note="EGFP, reverse primer" CDS 6106..6159 /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 6184..6783 /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(6184..6203) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6680..6700 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6791..6925 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6815..6834) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 6869..6888 /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind 7119..7152 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" primer_bind complement(7168..7185) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7168..7184) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7177..7199) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(7393..7412) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7512..7534 /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 acttcgaatt tgtatgcgtg cgcttgaagg 2341 gttgatcgga accttacaac agttgtagct atacggctgc gtgtggcttc taacgttatc 2401 catcgctaga agtgaaacga atgtgcgtag gtatatatat gaaatggagt tgctctctgc 2461 ttgggtcttc gagaagacct gttttagagc tagaaatagc aagttaaaat aaggctagtc 2521 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttgacact tttccattcc 2581 tcctgcttca tttgacgact tttttgccta cctggagcct gagagttgtt caataaaata 2641 aaaatgtttc gtttttttgc tttcgccagt atttattatt tttcatcaat atgtattcaa 2701 tttggtatgt atttagtaat tgtaatatat agacaatggt tttccgttga cgtacataca 2761 tctgacgtgt gtttatttag acataaggta cccagcatgc aattctatat tctaaaaaca 2821 caaatgatac ttctaaaaaa aaatcatgaa tggcatcaac tctgaatcaa atctttgcag 2881 atgcacctac ttctcatttc cactgtcaca tcatttttcc agatctcgct gcctgttatg 2941 tggcccacaa accaagacac gttttatggc cattaaagct ggctgatcgt cgccaaacac 3001 caaatacata atgaatatgt acacattcga gaaagaagcg atcaaagaag catcttcggg 3061 cggagtagga gaatgcggag gagaaggaga acgagctgat ctagtatctc tccacaatcc 3121 aatgccaact gaccaactgg ccatattcgg agcaatttga agccaatttc catcgcctgg 3181 cgatcgctcc attcttggct atatgttttt caccgttacc cggggccatt ttcaaagact 3241 cgtcggcaag ataagattgt gtcactcgct gtctctcttc atttgtcgaa gaatgctgag 3301 gaatttcgcg atgacgtcgg cgagtatttt gaagaatgag aataatttgt atttatacga 3361 aaatcagtta gtggaatttt ctacaaaaac atgttatcta tagataattt tgttgcaaaa 3421 tatgttgact atgacaaaga ttgtatgtat atacctttaa tgtattctca ttttcttatg 3481 tatttataat ggcaatgatg atactgatga tattttaaga tgatgccaga ccaaaaggct 3541 tgaatttctg cgtcttttgc cgaacgcagt gcatgtgcaa ttgttgtttt ttggaatatt 3601 caattttcgg actgtccgct ttgatttcag tttcttggct tattcaaaaa gcaaagtaaa 3661 gccaaaaaag cgagatggca ataccaaatg cggcaaaacg gtagtggaag gaaaggggtg 3721 cggggcagcg gaaggaaggg tggggcgggg cgtggcgggg tctgtggctg ggcgcgacgt 3781 caccgacgtt ggagccactc ctttgaccat gtgtgcgtgt gtgtattatt cgtgtctcgc 3841 cactcgccgg ttgttttttt ctttttatgc tgcgctctct ctagcgccat ctcgcttacg 3901 catgctcaac gcaccgcatg ttgccgtttc cttttatgcg tcattttggc tcgaaatagg 3961 caattattta aacaaagatt agtcaacgaa aacgctaaaa taaataagtc tacaatatgg 4021 ttacttattg ccatgtgtgt gcagccaacg atagcaacaa aagcaacaac acaggtggct 4081 ttccctcttt cactttttgt ttgcaagccg cgtgcgagca agacggcacg accggcaaac 4141 gcaattacgc tgacaaagag cagacgaagt tttggcgaaa aacatcaagg cgcctgatac 4201 gaatgcattt gcaataacaa ttgcgatatt taatattgtt tatgaagctg tttgacttca 4261 aaacacacaa aaaaaaaaat aaaacaaatt atttgaaaga gaattaggaa tcggacgctt 4321 atcgttaggg taacaacaag aaatgcttac tgagtcacag cctctggaaa actgccgcaa 4381 gccagagaga gagagaaaaa gagggagagc agcttagacc gcatgtgctt gtgtgtgagg 4441 cgtctctctc ttcgtctctg ttgcgcaaac gcatagactg cactgaaaaa atcgattacc 4501 tattttttat gaatgaatat ttgcactatt actattcaaa actattaaga tagcaatcac 4561 attcaatagc caaatactat accacctgag cgatgcaacg aaatgatcaa tttgagcaaa 4621 aatgctgcat atttaggacg gcatcattat agaaatgctt cttgctgtgt acttttctct 4681 cgtctggcag ctgtttcgcc gttattgtta aaaccggctt aagttaggtg tgttttctac 4741 gactagtgaa tgccctacta gaagatgtgt gttgcacaaa atgtccctgg aataaccaat 4801 ttgaagtgca gatagcagta aacgtaagct aatatgaata ttatttaact gtaatgtttt 4861 aatatcgctg gacattacta ataaacccac tataaacaca tgtacatatg tatgttttgg 4921 catacaatga gtagttgggg aaaaaatgtg taaaagcacc gtgaccatca cagcataaag 4981 ataaccagct gaagtatcga atatgagtaa cccccaaatt gaatcacatg ccgcaactga 5041 taggacccat ggaagtacac tcttcatggc gatatacaag acacacacaa gcacgaacac 5101 ccagttgcgg aggaaattct ccgtaaatga aaacccaatc ggcgaacaat tcatacccat 5161 atatggtaaa agttttgaac gcgacttgag agcggagagc attgcggctg ataaggtttt 5221 agcgctaagc gggctttata aaacgggctg cgggaccagt tttcatatca ctaccgtttg 5281 agttcttgtg ctgtgtggat actcctcccg acacaaagcc gctccatcag ccagcagtcg 5341 tctaatccag agaccccgga tccaggaatt cgcccttgtt taaacatggt gagcaagggc 5401 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 5461 cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg 5521 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 5581 agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc 5641 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 5701 acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa ccgcatcgag 5761 ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 5821 ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat caaggtgaac 5881 ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca ctaccagcag 5941 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct gagcacccag 6001 tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttccgc 6061 accgccgccg ggatcactct cggcatggac gagctgtaca agcttgaggg cagaggaagc 6121 cttctaacat gcggtgacgt ggaggagaat cccggccctg ctagcggtag cggcagcggt 6181 agcatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc 6241 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg 6301 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc 6361 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg 6421 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc 6481 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag 6541 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg 6601 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc 6661 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc 6721 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc 6781 tgactcgagg gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag 6841 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 6901 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 6961 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc 7021 tgattatgat cagtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt 7081 gggcgcgggg catgactatc gtcgccgcac ttatggtacg cgcccgggga gcccaagggc 7141 acgccctggc acccgaaggg cgaattcact ggccgtcgtt ttacaacgtc gtgactggga 7201 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 7261 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 7321 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 7381 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 7441 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 7501 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 7561 ga //