LOCUS Exported 7589 bp ds-DNA circular SYN 20-APR-2022 DEFINITION 3rd gen lentiviral backbone for cloning and expression of new shRNA sequences. Uses neomycin for selection.. ACCESSION . VERSION . KEYWORDS pLKO.1 neo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7589) TITLE Stewart Lab Plasmids REFERENCE 2 (bases 1 to 7589) AUTHORS . TITLE Direct Submission JOURNAL Exported Apr 20, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7589 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 426..444 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 880..1674 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(934..953) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1544..1563 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(1696..1712) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1697..1713) /label=pBluescriptKS /note="For pBluescript vector" LTR 1795..2028 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2100..2221 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2137..2156) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2191..2210 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 2261..2396 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2323..2342 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2416..2435) /label=T7 /note="T7 promoter, forward primer" promoter complement(2417..2435) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2445..2462) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2445..2461) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2454..2476) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2603..3058 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2690..2709) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2900..2921 /label=F1ori-F /note="F1 origin, forward primer" promoter 3084..3188 /gene="bla" /label=AmpR promoter CDS 3189..4049 /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(3407..3426) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4220..4808 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4709..4728 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4962..4979 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5096..5117 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5132..5162 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5170..5186 /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 5175..5197 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5194..5210 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5194..5210 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5229..5249 /label=T3 /note="T3 promoter, forward primer" promoter 5231..5249 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 5277..5503 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 5504..5684 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5731..5856 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 6349..6582 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6767..6811 /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 7109..7349 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7109..7129 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7280..7299 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 7420..7537 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 7586..507 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" ORIGIN 1 ttggggttgc 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 atcaagcttc acgctgccgc 541 aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag 601 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 661 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 721 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 781 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 841 tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 901 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 961 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 1021 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 1081 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 1141 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 1201 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 1261 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 1321 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 1381 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 1441 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 1501 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 1561 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 1621 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 1681 ctctggggtt cgcatcgata ccgtcgacct cgaggggggg cccggtacct ttaagaccaa 1741 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 1801 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt 1861 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 1921 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 1981 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat 2041 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 2101 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 2161 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 2221 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc 2281 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 2341 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 2401 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 2461 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 2521 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 2581 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 2641 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 2701 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 2761 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 2821 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 2881 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 2941 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 3001 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 3061 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 3121 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 3181 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 3241 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 3301 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 3361 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 3421 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 3481 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 3541 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 3601 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 3661 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 3721 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 3781 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 3841 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 3901 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 3961 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 4021 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 4081 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 4141 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 4201 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 4261 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 4321 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 4381 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 4441 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 4501 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 4561 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 4621 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 4681 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 4741 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 4801 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 4861 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 4921 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 4981 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 5041 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 5101 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 5161 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 5221 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta 5281 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt 5341 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc 5401 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca 5461 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga 5521 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 5581 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 5641 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg 5701 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa 5761 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc 5821 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga 5881 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat 5941 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca 6001 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa 6061 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag 6121 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc 6181 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg 6241 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 6301 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt 6361 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 6421 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat 6481 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 6541 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc 6601 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 6661 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac 6721 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga 6781 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct 6841 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt 6901 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac 6961 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga 7021 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gatcacgaga 7081 ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt 7141 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa 7201 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 7261 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 7321 cttggcttta tatatcttgt ggaaaggacg aaacaccggt ccgcaggtat gcacgcgtga 7381 attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 7441 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 7501 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 7561 agatccactt tggccgcggc tcgaggggg //