LOCUS Exported 8648 bp ds-DNA circular SYN 08-SEP-2021 DEFINITION CRISPR gRNA expression vector with an improved scaffold and puro/BFP markers. ACCESSION . VERSION . KEYWORDS pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8648) 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 8648) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 8, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8648 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 243..441 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 396..416 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 459..639 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 686..811 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1310..1543 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1728..1772 /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 2070..2187 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2262..2502 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2262..2282 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2433..2452 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 2637..3136 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(2672..2695) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3069..3088 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3176..3772 /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(3176..3195) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3672..3692 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 3791..3844 /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 3845..4546 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" primer_bind complement(4606..4626) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5024..5035) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5487..5504) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5493..5717 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5743..5763) /label=T3 /note="T3 promoter, forward primer" promoter complement(5743..5761) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5782..5798) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5782..5798) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5795..5817) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5806..5822 /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(5830..5860) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5875..5896 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6013..6030) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6184..6772) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6264..6283) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6943..7803) /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 7566..7585 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7804..7908) /gene="bla" /label=AmpR promoter rep_origin complement(7934..8389) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8071..8092) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8283..8302 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8516..8538 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8530..8547 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8531..8547 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 8557..8576 /label=T7 /note="T7 promoter, forward primer" promoter 8557..8575 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 61 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 121 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 181 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 241 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt 301 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 361 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg 421 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga 481 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 541 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 601 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt 661 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg 721 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg 781 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg 841 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt 901 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga 961 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact 1021 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa 1081 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc 1141 acagcaagcg gccggccgcg ctgatcttca gacctggagg aggagatatg agggacaatt 1201 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca 1261 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt 1321 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg 1381 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta 1441 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa 1501 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct 1561 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc 1621 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca 1681 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag 1741 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc 1801 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt 1861 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga 1921 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag 1981 agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt gcgccaattc 2041 tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac 2101 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa 2161 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccagtt 2221 tggttagtac cgggccctac gcgtccaagg tcgggcagga agagggccta tttcccatga 2281 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga 2341 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt 2401 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa 2461 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtcttcga 2521 gaagacacgt ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg 2581 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tggatccaat tctaccgggt 2641 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac 2701 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca 2761 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa 2821 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct 2881 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg 2941 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg 3001 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg 3061 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc 3121 atctccgggc ctttcgacct gcatccatct agatctcgag cagctgaagc ttaccatgac 3181 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac 3241 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca 3301 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg 3361 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt 3421 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg 3481 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc 3541 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc 3601 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac 3661 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt 3721 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggcggcgg 3781 gtccggagga gagggcagag gaagtctcct aacatgcggt gacgtggagg agaatcctgg 3841 cccaatgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt 3901 ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca 3961 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc 4021 tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt 4081 cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaggacgg 4141 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt 4201 caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg 4261 ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga 4321 catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag 4381 atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact 4441 ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc 4501 cagatactgc gacctcccta gcaaactggg gcacaagctt aattgagcgg ccgctcaacc 4561 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 4621 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 4681 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 4741 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 4801 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 4861 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 4921 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 4981 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 5041 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc tacgccttcg 5101 ccctcagacg agtcggatct ccctttgggc cgcctccccg caggtacctt taagaccaat 5161 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 5221 gctaattcac tcccaaagaa gtcaagatct gctttttgcc tgtactgggt ctctctggtt 5281 agaccagagt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg 5341 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt 5401 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc 5461 tctagcagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5521 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5581 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5641 tggggtgggg caggacagca agggggagga ttgggaagtc aatagcaggc atgctgggga 5701 tgcggtgggc tctatgggcg gccgttaatt aagcttttgt tccctttagt gagggttaat 5761 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 5821 aattccacac aacatacgag ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt 5881 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5941 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 6001 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 6061 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 6121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 6181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 6241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 6301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 6361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 6421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 6481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 6541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 6601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 6661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 6721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 6781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 6841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 6901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 6961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 7021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 7081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 7141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 7201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 7261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 7321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 7381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 7441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 7501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 7561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 7621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 7681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 7741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 7801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 7861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 7921 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt 7981 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 8041 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 8101 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 8161 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 8221 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 8281 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 8341 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca 8401 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 8461 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 8521 tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa 8581 ttgactagta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 8641 ccattgac //