LOCUS Exported 7396 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral Vector for Sp sgRNA delivery without SpCas9, tagRFP657, EFS Promoter driven. ACCESSION . VERSION . KEYWORDS pLKO5.sgRNA.EFS.tRFP657 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7396) 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 7396) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7396 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 5..231 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 232..412 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 459..584 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1077..1310 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1495..1539 /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 1717..1957 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1717..1737 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1888..1907 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1984..2059 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2107..2224 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2293..2504 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 2530..3231 /codon_start=1 /product="far-red monomeric fluorescent protein with an emission peak at 657 nm (Morozova et al., 2010)" /label=TagRFP657 /note="derived from mKate" /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC DLPSKLGHKLN" misc_feature 3253..3841 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3306..3326) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3724..3735) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3913..4146 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4224..4345 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4261..4280) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4315..4334 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4385..4520 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4447..4466 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4540..4559) /label=T7 /note="T7 promoter, forward primer" promoter complement(4541..4559) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4569..4586) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4569..4585) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4578..4600) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4727..5182 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4814..4833) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5024..5045 /label=F1ori-F /note="F1 origin, forward primer" promoter 5208..5312 /gene="bla" /label=AmpR promoter CDS 5313..6173 /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(5531..5550) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6344..6932 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6833..6852 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7086..7103 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7220..7241 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7256..7286 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7294..7310 /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 7299..7321 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7318..7334 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7318..7334 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7353..7373 /label=T3 /note="T3 promoter, forward primer" promoter 7355..7373 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc 241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct 1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta 1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgtgaccgtc 1981 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 2041 agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat 2101 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 2161 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 2221 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg 2281 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 2341 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 2401 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 2461 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga 2521 tctgccacca tgagcgagct gatcaccgag aacatgcaca tgaagctgta catggagggc 2581 accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc 2641 acccagaccc agagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc 2701 ctggctacca gcttcatgta cggcagtcac accttcatca accacaccca gggcatcccc 2761 gacttctgga agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa 2821 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac 2881 aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca 2941 ctcggctggg aggcccacac cgagatgctg taccccgctg acggcggcct ggaaggcaga 3001 accgcgctgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt caagaccaca 3061 tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggactac 3121 agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct 3181 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta aggtaccacg 3241 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 3301 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 3361 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 3421 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 3481 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 3541 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 3601 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 3661 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 3721 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 3781 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 3841 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag 3901 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt 3961 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga 4021 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc 4081 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct 4141 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga 4201 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa 4261 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 4321 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc 4381 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 4441 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 4501 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac 4561 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 4621 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 4681 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc 4741 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 4801 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 4861 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 4921 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 4981 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 5041 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 5101 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 5161 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta 5221 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 5281 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 5341 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 5401 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 5461 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 5521 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 5581 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 5641 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 5701 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 5761 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 5821 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 5881 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 5941 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 6001 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 6061 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 6121 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 6181 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 6241 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 6301 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 6361 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 6421 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 6481 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 6541 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 6601 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 6661 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 6721 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 6781 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 6841 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 6901 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 6961 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 7021 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 7081 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc 7141 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 7201 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 7261 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 7321 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa 7381 agctggagct gcaagc //