LOCUS Exported 8649 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 8649) 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 8649) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 8, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8649 /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..3137 /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 3070..3089 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3177..3773 /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(3177..3196) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3673..3693 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 3792..3845 /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 3846..4547 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" primer_bind complement(4607..4627) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5025..5036) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5488..5505) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5494..5718 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5744..5764) /label=T3 /note="T3 promoter, forward primer" promoter complement(5744..5762) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5783..5799) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5783..5799) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5796..5818) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5807..5823 /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(5831..5861) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5876..5897 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6014..6031) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6185..6773) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6265..6284) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6944..7804) /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 7567..7586 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7805..7909) /gene="bla" /label=AmpR promoter rep_origin complement(7935..8390) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8072..8093) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8284..8303 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8517..8539 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8531..8548 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8532..8548 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 8558..8577 /label=T7 /note="T7 promoter, forward primer" promoter 8558..8576 /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 gtccgggggn nnggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg 3061 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct 3121 catctccggg cctttcgacc tgcatccatc tagatctcga gcagctgaag cttaccatga 3181 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca 3241 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc 3301 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg 3361 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg 3421 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc 3481 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg 3541 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg 3601 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga 3661 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg 3721 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gccggcggcg 3781 ggtccggagg agagggcaga ggaagtctcc taacatgcgg tgacgtggag gagaatcctg 3841 gcccaatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg 3901 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc 3961 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg 4021 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact 4081 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaggacg 4141 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg 4201 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg 4261 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg 4321 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata 4381 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac 4441 tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg 4501 ccagatactg cgacctccct agcaaactgg ggcacaagct taattgagcg gccgctcaac 4561 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 4621 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 4681 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 4741 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 4801 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 4861 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 4921 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 4981 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 5041 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt ctacgccttc 5101 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcaggtacct ttaagaccaa 5161 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 5221 ggctaattca ctcccaaaga agtcaagatc tgctttttgc ctgtactggg tctctctggt 5281 tagaccagag tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg 5341 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg 5401 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat 5461 ctctagcagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 5521 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 5581 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 5641 gtggggtggg gcaggacagc aagggggagg attgggaagt caatagcagg catgctgggg 5701 atgcggtggg ctctatgggc ggccgttaat taagcttttg ttccctttag tgagggttaa 5761 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 5821 caattccaca caacatacga gccgggagca taaagtgtaa agcctggggt gcctaatgag 5881 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5941 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 6001 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 6061 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 6121 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 6181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 6241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 6301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 6361 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 6421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 6481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 6541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 6601 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 6661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 6721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 6781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 6961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 7021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 7081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 7141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 7201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 7261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 7321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 7381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 7441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 7501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 7561 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 7621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 7681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 7741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 7801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 7861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 7921 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg 7981 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 8041 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8101 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8161 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8221 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8281 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct 8341 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc 8401 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 8461 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 8521 gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga 8581 attgactagt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 8641 cccattgac //