LOCUS Exported 8757 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cas9 activity reporter with mCherry and BFP. ACCESSION . VERSION . KEYWORDS pKLV2-U6gRNA5(gBFP)-PGKmCherry2ABFP-W SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8757) AUTHORS Tzelepis K, Koike-Yusa H, De Braekeleer E, Li Y, Metzakopian E, Dovey OM, Mupo A, Grinkevich V, Li M, Mazan M, Gozdecka M, Ohnishi S, Cooper J, Patel M, McKerrell T, Chen B, Domingues AF, Gallipoli P, Teichmann S, Ponstingl H, McDermott U, Saez-Rodriguez J, Huntly BJ, Iorio F, Pina C, Vassiliou GS, Yusa K TITLE A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia. JOURNAL Cell Rep. 2016 Oct 18;17(4):1193-1205. doi: 10.1016/j.celrep.2016.09.079. PUBMED 27760321 REFERENCE 2 (bases 1 to 8757) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8757 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 960..1158 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1113..1133 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 1176..1356 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1403..1528 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 2027..2260 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2445..2489 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 2787..2904 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2979..3219 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2979..2999 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3150..3169 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 3356..3855 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(3391..3414) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3788..3807 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3885..4595 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MSVSKGEEDNSAIIKEFLRFKVHMEGSVNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGAL KGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYER AEGRHSTGGMDELYK" primer_bind complement(4033..4051) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 4301..4320 /label=mCherry-F /note="mCherry, forward primer" CDS 4614..4667 /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 4668..5372 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MGSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL KLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY CDLPSKLGHKLN" primer_bind complement(5432..5452) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5850..5861) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6313..6330) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6319..6543 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6569..6589) /label=T3 /note="T3 promoter, forward primer" promoter complement(6569..6587) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6608..6624) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6608..6624) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6621..6643) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6632..6648 /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(6656..6686) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6701..6722 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6839..6856) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7010..7598) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7090..7109) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7769..8629) /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 8392..8411 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8630..8734) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgactagtac 661 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 721 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 781 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 841 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 901 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 961 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1021 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1081 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1141 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct 1201 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 1261 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 1321 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 1381 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 1441 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 1501 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 1561 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 1621 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 1681 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1741 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1801 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1861 gcaagcggcc ggccgcgctg atcttcagac ctggaggagg agatatgagg gacaattgga 1921 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca 1981 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc 2041 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac 2101 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg 2161 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa 2221 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg 2281 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg 2341 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa 2401 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat 2461 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt 2521 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg 2581 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc 2641 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga 2701 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc 2761 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 2821 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 2881 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg 2941 ttagtaccgg gccctacgcg tccaaggtcg ggcaggaaga gggcctattt cccatgattc 3001 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg 3061 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 3121 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 3181 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgag cacgcccccg 3241 tcctcgtgtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt 3301 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ggatccaatt ctaccgggta 3361 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact 3421 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa 3481 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag 3541 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc 3601 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc 3661 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg 3721 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga 3781 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca 3841 tctccgggcc tttcgacctg catccatcta gatctcgagc caccatgagc gtgagcaagg 3901 gcgaggagga taactccgcc atcatcaagg agttcctgcg cttcaaggtg cacatggagg 3961 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg 4021 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca 4081 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc 4141 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg 4201 aggacggcgg cgtggtgacc gtgacccagg actcctctct gcaggacggc gagttcatct 4261 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga 4321 caatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg 4381 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca 4441 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg 4501 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc 4561 gccactccac cggcggcatg gacgagctgt ataagggcgg cgggtccgga ggagagggca 4621 gaggatctct tctaacatgc ggtgacgtgg aggagaatcc tggcccaatg ggcagcgagc 4681 tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact 4741 tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca 4801 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct 4861 acggcagcaa gaccttcatc aaccacaccc aggggatccc cgacttcttc aagcagtcct 4921 tccccgaggg cttcacatgg gagagggtca ccacctacga ggacgggggc gtgctgaccg 4981 ccacccagga caccagcctc caggacgggt gcctcatcta caacgtcaag atcaggggcg 5041 tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca 5101 ccgagaccct gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc 5161 tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg 5221 ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg 5281 aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc 5341 tccctagcaa actggggcac aagcttaatt gagcggccgc tcaacctctg gattacaaaa 5401 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 5461 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 5521 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 5581 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 5641 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 5701 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 5761 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 5821 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 5881 gcggcctgct gccggctctg cggcctcttc cgcgtctacg ccttcgccct cagacgagtc 5941 ggatctccct ttgggccgcc tccccgcagg tacctttaag accaatgact tacaaggcag 6001 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 6061 aaagaagtca agatctgctt tttgcctgta ctgggtctct ctggttagac cagagtctct 6121 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 6181 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 6241 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtttaaa 6301 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 6361 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 6421 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 6481 acagcaaggg ggaggattgg gaagtcaata gcaggcatgc tggggatgcg gtgggctcta 6541 tgggcggccg ttaattaagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt 6601 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 6661 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 6721 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6781 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6841 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6901 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6961 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 7021 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 7081 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 7141 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 7201 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 7261 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 7321 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 7381 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 7441 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 7501 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 7561 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 7621 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 7681 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7741 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7801 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7861 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7921 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7981 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 8041 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 8101 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 8161 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 8221 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 8281 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 8341 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 8401 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 8461 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 8521 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 8581 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 8641 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 8701 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac //