LOCUS Exported 7539 bp ds-DNA circular SYN 15-JUN-2022 DEFINITION 3rd gen lentiviral backbone for cloning and expression of new shRNA sequences. Uses hygromycin for selection.. ACCESSION . VERSION . KEYWORDS pLKO.1 hygro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7539) TITLE empty vector REFERENCE 2 (bases 1 to 7539) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 15, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7539 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 240..260 /label=T3 /note="T3 promoter, forward primer" promoter 242..260 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 288..514 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 515..695 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 742..867 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1360..1593 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1778..1822 /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 2120..2360 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2120..2140 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2291..2310 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2431..2548 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2597..3107 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 3026..3044 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 3197..4222 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" primer_bind complement(4246..4262) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4247..4263) /label=pBluescriptKS /note="For pBluescript vector" LTR 4345..4578 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4650..4771 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4687..4706) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4741..4760 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4811..4946 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4873..4892 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4966..4985) /label=T7 /note="T7 promoter, forward primer" promoter complement(4967..4985) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4995..5012) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4995..5011) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5004..5026) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5153..5608 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5240..5259) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5450..5471 /label=F1ori-F /note="F1 origin, forward primer" promoter 5634..5738 /gene="bla" /label=AmpR promoter CDS 5739..6599 /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(5957..5976) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6770..7358 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7259..7278 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7512..7529 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg 241 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc 301 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga 361 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg 421 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga 481 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg 541 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 601 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 661 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa 721 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg 781 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag 841 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg 901 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg 961 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc 1021 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga 1081 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac 1141 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag 1201 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga 1261 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa 1321 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt 1381 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag 1441 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca 1501 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc 1561 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact 1621 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat 1681 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat 1741 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga 1801 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat 1861 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact 1921 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc 1981 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag 2041 agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg 2101 agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg 2161 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt 2221 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg 2281 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt 2341 atatatcttg tggaaaggac gaaacaccgg tccgcaggta tgcacgcgtg aattctcgac 2401 ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta 2461 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 2521 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact 2581 ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc 2641 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc 2701 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc 2761 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt 2821 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc 2881 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag 2941 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag 3001 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt 3061 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg 3121 atcaagcttg ccagctgggg cgccctctgg taaggttggg aagccctgca gatctgatcc 3181 cggggggcaa tgagatatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct 3241 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg 3301 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga 3361 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc 3421 ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct cccgccgtgc 3481 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt 3541 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc 3601 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc 3661 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc 3721 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt 3781 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat 3841 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg 3901 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga 3961 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta 4021 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc 4081 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc 4141 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac 4201 tcgtccgagg gcaaaggaat agagtagatg ccgaccggga tctatcgata ccgtcgacct 4261 cgaggggggg cccggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc 4321 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc 4381 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 4441 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 4501 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 4561 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg 4621 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca 4681 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 4741 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct 4801 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 4861 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 4921 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg 4981 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 5041 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 5101 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 5161 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 5221 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 5281 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 5341 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 5401 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 5461 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 5521 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 5581 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 5641 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 5701 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 5761 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 5821 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 5881 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 5941 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 6001 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 6061 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 6121 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 6181 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 6241 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 6301 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 6361 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 6421 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 6481 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 6541 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 6601 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 6661 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 6721 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 6781 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 6841 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 6901 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 6961 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 7021 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 7081 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 7141 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 7201 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 7261 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 7321 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 7381 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 7441 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 7501 cgaccgagcg cagcgagtca gtgagcgagg aagcggaag //