LOCUS Exported 8919 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Backbone for lentiviral gene silencing with monomeric Kusabira-Orange2 expression. ACCESSION . VERSION . KEYWORDS pLKO.1-TRC.mKO2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8919) AUTHORS Dias TITLE Transcriptomic data of triple negative breast cancer revealed GBP1 as potential new therapeutic target JOURNAL Unpublished REFERENCE 2 (bases 1 to 8919) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8919 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 426..444 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 519..1172 /codon_start=1 /product="monomeric Kusabira-Orange 2 fluorescent protein" /label=mKO2 /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS" LTR 1274..1507 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 1579..1700 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1616..1635) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1670..1689 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 1740..1875 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1802..1821 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(1895..1914) /label=T7 /note="T7 promoter, forward primer" promoter complement(1896..1914) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1924..1941) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1924..1940) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1933..1955) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2082..2537 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2169..2188) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2379..2400 /label=F1ori-F /note="F1 origin, forward primer" promoter 2563..2667 /gene="bla" /label=AmpR promoter CDS 2668..3528 /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(2886..2905) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3699..4287 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4188..4207 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4441..4458 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4575..4596 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4611..4641 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4649..4665 /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 4654..4676 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4673..4689 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4673..4689 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4708..4728 /label=T3 /note="T3 promoter, forward primer" promoter 4710..4728 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 4756..4982 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 4983..5163 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5210..5335 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5828..6061 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6246..6290 /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 6588..6828 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 6588..6608 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 6759..6778 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 8750..8867 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 8916..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 gatctaccat ggtgagcgtg atcaagcccg 541 agatgaagat gaggtactac atggacggct ccgtcaatgg gcatgagttc acaatcgagg 601 gtgagggcac aggcagacct tacgagggac atcaggagat gacactgcgc gtcacaatgg 661 ccgagggcgg gccaatgcct ttcgccttcg acctggtgtc ccacgtgttc tgttacggcc 721 acagagtttt taccaagtac ccagaagaga tcccagacta tttcaagcag gcctttcctg 781 agggcctgtc ctgggagagg tccctggagt tcgaggacgg cggctccgcc tccgtgagcg 841 cccacatcag cctgaggggc aacaccttct accacaagtc caagttcacc ggcgtgaact 901 tccccgccga cggccccatc atgcagaacc agagcgtgga ctgggagccc tccaccgaga 961 agatcaccgc cagcgacggc gtgctgaagg gcgacgtgac catgtacctg aagctggagg 1021 gcggcggcaa ccacaagtgc cagatgaaga ccacctacaa ggccgccaag gagatcctgg 1081 agatgcccgg cgaccactac atcggccaca ggctggtgag gaagaccgag ggcaacatca 1141 ccgagcaggt ggaggacgcc gtggcccact ccggtggcgg aggctcgggc ggaggtgggt 1201 cgggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa 1261 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct 1321 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg 1381 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt 1441 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc 1501 tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga 1561 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat 1621 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 1681 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca 1741 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 1801 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag 1861 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg 1921 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 1981 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 2041 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 2101 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 2161 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 2221 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 2281 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 2341 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 2401 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 2461 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 2521 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 2581 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 2641 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 2701 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 2761 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 2821 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2881 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 2941 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 3001 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 3061 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 3121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 3181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 3241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 3301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 3361 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 3421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 3481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 3541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 3601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 3661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 3721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 3781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 3841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 3901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 3961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 4021 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 4081 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 4141 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 4201 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 4261 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 4321 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 4381 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 4441 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg 4501 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 4561 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 4621 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 4681 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg 4741 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga 4801 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag 4861 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac 4921 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa 4981 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 5041 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 5101 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 5161 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg 5221 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac 5281 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat 5341 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa 5401 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa 5461 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc 5521 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg 5581 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga 5641 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag 5701 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac 5761 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag 5821 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag 5881 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga 5941 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca 6001 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc 6061 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 6121 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca 6181 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc 6241 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt 6301 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg 6361 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt 6421 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa 6481 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatctc 6541 gacggtatcg atcacgagac tagcctcgag cggccgcccc cttcaccgag ggcctatttc 6601 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta 6661 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc 6721 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa 6781 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggtg 6841 tgttgtaaat gagcacacaa aatacacatg ctaaaatatt atattctatg acctttataa 6901 aatcaaccaa aatcttcttt ttaataactt tagtatcaat aattagaatt tttatgttcc 6961 tttttgcaaa cttttaataa aaatgagcaa aataaaaaaa cgctagtttt agtaactcgc 7021 gttgttttct tcacctttaa taatagctac tccaccactt gttcctaagc ggtcagctcc 7081 tgcttcaatc attttttgag catcttcaaa tgttctaact ccaccagctg ctttaactaa 7141 agcattgtct ttaacaactg acttcattag tttaacatct tcaaatgttg cacctgattt 7201 tgaaaatcct gttgatgttt taacaaattc taatccagct tcaacagcta tttcacaagc 7261 tttcatgatt tcttcttttg ttaataaaca attttccata atacatttaa caacatgtga 7321 tccagctgct ttttttacag ctttcatgtc ttctaaaact aattcataat ttttgtcttt 7381 taatgcacca atatttaata ccatatcaat ttctgttgca ccatctttaa ttgcttcaga 7441 aacttcgaat gcttttgtag ctgttgtgca tgcacctaga ggaaaaccta caacatttgt 7501 tattcctaca tttgtgcctt ttaataattc tttacaatag cttgttcaat atgaattaac 7561 acaaactgtt gcaaaatcaa attcaattgc ttcatcacat aattgtttaa tttcagcttt 7621 cgtagcatct tgttttaata atgtgtgatc tatatatttg tttagtttca ttttttctcc 7681 tatatattca tttttaattt taattcttta ataatttcgt ctactttaac tttagcgttt 7741 tgaacagatt caccaacacc tataaaataa atttttagtt taggttcagt tccacttggg 7801 cgaacagcaa atcatgactt atcttctaaa taaaatttta gtaagtcttg tcctggcata 7861 ttatacattc catcgatgta gtcttcaaca ttaacaactt taagtccagc aatttgagtt 7921 aagggtgttg ctctcaatga tttcattaat ggttcaattt ttaatttctt ttcttctggt 7981 ttaaaattca agtttaaagt gaaagtgtaa tatgcaccca tttctttaaa taaatcttct 8041 aaatagtcta ctaatgtttt attttgtttt ttataaaatc aagcagcctc tgctattaat 8101 atagaagctt gtattccatc tttatctcta gctgagtcat caattacata tccataactt 8161 tcttcataag caaaaacaaa atttaatccg ttatcttctt ctttagcaat ttctctaccc 8221 attcatttaa atccagttaa agtttttaca atattaactc catatttttc atgagcgatt 8281 ctatcaccca aatcacttgt tacaaaactt gaatatagag ccggattttt tggaatgcta 8341 tttaagcgtt ttagatttga taattttcaa tcaattaaaa ttggtcctgt ttgatttcca 8401 tctaatctta caaaatgacc atcatgtttt attgccattc caaatctgtc agcatctggg 8461 tcattcataa taataatatc tgcatcatgt ttaataccat attcaagcgg tatttttcat 8521 gcaggatcaa attctggatt tggatttaca acatttttaa atgtttcatc ttcaaatgca 8581 tgctcttcaa cctcaataac gttatatcct gattcacgta atatttttgg ggtaaattta 8641 gttcctgttc cattaactgc gctaaaaata atttttaaat cttttttagc ttcttgctct 8701 tttttgtaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa 8761 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 8821 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 8881 gggacagcag agatccactt tggccgcggc tcgaggggg //