LOCUS Exported 7288 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral Vector for Sp sgRNA delivery without SpCas9, Puromycin resistance, EFS Promoter driven. ACCESSION . VERSION . KEYWORDS pLKO5.sgRNA.EFS.PAC SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7288) AUTHORS Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL TITLE Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. JOURNAL Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951. PUBMED 24952903 REFERENCE 2 (bases 1 to 7288) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7288 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 6..232 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 233..413 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 460..585 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1078..1311 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1496..1540 /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 1718..1958 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1718..1738 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1889..1908 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1985..2060 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2108..2225 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2294..2505 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 2531..3130 /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(2531..2550) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3027..3047 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 3146..3734 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3199..3219) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3617..3628) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3806..4039 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4117..4238 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4154..4173) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4208..4227 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4278..4413 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4340..4359 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4433..4452) /label=T7 /note="T7 promoter, forward primer" promoter complement(4434..4452) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4462..4479) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4462..4478) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4471..4493) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4620..5075 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4707..4726) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4917..4938 /label=F1ori-F /note="F1 origin, forward primer" promoter 5101..5205 /gene="bla" /label=AmpR promoter CDS 5206..6066 /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(5424..5443) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6237..6825 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6726..6745 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6979..6996 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7113..7134 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7149..7179 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7187..7203 /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 7192..7214 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7211..7227 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7211..7227 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7246..7266 /label=T3 /note="T3 promoter, forward primer" promoter 7248..7266 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg 481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt 541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa 901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag 961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt 1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg 1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac 1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct 1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct 1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg 1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa 1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa 1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa 1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac 1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag 1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc 1681 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc 1741 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt 1801 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt 1861 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta 1921 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag acgtgaccgt 1981 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 2041 aagtggcacc gagtcggtgc ttttttgaat tctagatctt gagacaaatg gcagtattca 2101 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag 2161 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa 2221 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagtggctcc 2281 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg 2341 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc 2401 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc 2461 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg 2521 atccgccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc 2581 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt 2641 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt 2701 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 2761 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga 2821 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg 2881 gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa 2941 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc 3001 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac 3061 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc 3121 cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt 3181 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 3241 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 3301 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 3361 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 3421 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 3481 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 3541 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc 3601 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 3661 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 3721 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc 3781 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat 3841 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 3901 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 3961 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 4021 gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta 4081 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat 4141 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 4201 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc 4261 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 4321 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat 4381 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag 4441 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 4501 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 4561 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga 4621 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 4681 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 4741 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 4801 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 4861 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 4921 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 4981 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 5041 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg 5101 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 5161 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 5221 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 5281 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 5341 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 5401 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 5461 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 5521 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 5581 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 5641 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 5701 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 5761 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 5821 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 5881 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 5941 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 6001 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 6061 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 6121 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 6181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 6241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 6301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 6361 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 6421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 6481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 6541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 6601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 6661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 6721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 6901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 6961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 7021 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 7081 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 7141 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 7201 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac 7261 taaagggaac aaaagctgga gctgcaag //