LOCUS Exported 7566 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION lentiviral vector expressing sgRNA targeting LacZ. ACCESSION . VERSION . KEYWORDS pLCKO_LacZ_sgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7566) AUTHORS Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, Mis M, Zimmermann M, Fradet-Turcotte A, Sun S, Mero P, Dirks P, Sidhu S, Roth FP, Rissland OS, Durocher D, Angers S, Moffat J TITLE High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. JOURNAL Cell. 2015 Dec 3;163(6):1515-26. doi: 10.1016/j.cell.2015.11.015. Epub 2015 Nov 25. PUBMED 26627737 REFERENCE 2 (bases 1 to 7566) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7566 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 7..507 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 426..444 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 529..1128 /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(529..548) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1025..1045 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 1144..1732 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1197..1217) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(1615..1626) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 1804..2037 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2115..2236 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2152..2171) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2206..2225 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 2276..2411 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2338..2357 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2431..2450) /label=T7 /note="T7 promoter, forward primer" promoter complement(2432..2450) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2460..2477) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2460..2476) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2469..2491) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2618..3073 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2705..2724) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2915..2936 /label=F1ori-F /note="F1 origin, forward primer" promoter 3099..3203 /gene="bla" /label=AmpR promoter CDS 3204..4064 /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(3422..3441) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4235..4823 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4724..4743 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4977..4994 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5111..5132 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5147..5177 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5185..5201 /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 5190..5212 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5209..5225 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5209..5225 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5244..5264 /label=T3 /note="T3 promoter, forward primer" promoter 5246..5264 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 5292..5518 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 5519..5699 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5746..5871 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 6364..6597 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6782..6826 /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" promoter 7004..7244 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7004..7024 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7175..7194 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 7274..7349 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 7397..7514 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 cccgggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 481 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac 541 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc 601 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag 661 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg 721 tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag cgtcgaagcg 781 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc 841 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc 901 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg 961 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 1021 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 1081 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc 1141 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 1201 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 1261 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 1321 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 1381 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 1441 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 1501 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg 1561 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 1621 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 1681 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt 1741 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 1801 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 1861 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 1921 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 1981 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 2041 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat 2101 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 2161 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 2221 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc 2281 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 2341 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 2401 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 2461 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 2521 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 2581 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 2641 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 2701 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 2761 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 2821 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 2881 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 2941 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 3001 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 3061 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 3121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 3181 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 3241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 3301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 3361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 3421 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 3481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 3541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 3601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 3661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 3721 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 3781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 3841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 3901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 3961 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 4021 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 4081 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 4141 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4201 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4261 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4321 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4381 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 4441 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4501 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 4561 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 4621 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 4681 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 4741 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 4801 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 4861 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 4921 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 4981 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 5041 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 5101 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 5161 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 5221 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc 5281 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag 5341 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag 5401 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac 5461 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg 5521 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 5581 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 5641 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 5701 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg 5761 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca 5821 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag 5881 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa 5941 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct 6001 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt 6061 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg 6121 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa 6181 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga 6241 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt 6301 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg 6361 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc 6421 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa 6481 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa 6541 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg 6601 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg 6661 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga 6721 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga 6781 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa 6841 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg 6901 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc 6961 attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat 7021 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt 7081 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg 7141 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg 7201 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaggta ccgcccgaat 7261 ctctatcgtg cgggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc 7321 aacttgaaaa agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg 7381 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa 7441 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa 7501 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg 7561 aggggg //