LOCUS Exported 8985 bp ds-DNA circular SYN 28-NOV-2016 DEFINITION pLK-TetON-ministuffer, with the AgeI site mutated to a NheI. ACCESSION . VERSION . KEYWORDS EZ-Tet-pLKO-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8985) AUTHORS . TITLE Direct Submission JOURNAL Exported Monday, Nov 28, 2016 from SnapGene Viewer 3.2.1 http://www.snapgene.com COMMENT Modified from original pLK-Puro-TetON construct by adding EcoRI site and shrinking the stuffer region down to about 200bp. Previously called pLKO-TetON-Puro(NheI-EcorI) FEATURES Location/Qualifiers source 1..8985 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 452..547 /note="H1/TetO Promoter" misc_feature 555..775 /note="Stuffer" misc_feature 817..934 /note="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1" promoter 983..1493 /note="hPGK promoter" /note="human phosphoglycerate kinase 1 promoter" intron 1502..2074 /note="beta-globin intron" /note="intron from rabbit beta-globin gene" promoter 2129..2147 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" CDS 2158..2775 /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /note="TetR" /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG" misc_feature 2813..3386 /note="IRES" CDS 3406..4005 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /note="PuroR" /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" LTR 4133..4366 /note="3' LTR (Delta-U3)" /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4438..4559 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin 4599..4734 /note="SV40 ori" /note="SV40 origin of replication" promoter complement(4755..4773) /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4783..4799) /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" rep_origin 4941..5396 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 5422..5526 /gene="bla" /note="AmpR promoter" CDS 5527..6387 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 6558..7146 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 7434..7455 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding site" /note="CAP binding activates transcription in the presence of cAMP." promoter 7470..7500 /note="lac promoter" /note="promoter for the E. coli lac operon" protein_bind 7508..7524 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /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 7532..7548 /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" promoter 7569..7587 /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" promoter 7615..7841 /note="RSV promoter" /note="Rous sarcoma virus enhancer/promoter" LTR 7842..8022 /note="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 8069..8194 /note="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 8687..8920 /note="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." ORIGIN 1 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa 61 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa 121 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca 181 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt 241 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat 301 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag 361 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt 421 atcgatcacg agactagcct cgagcggccg caatatttgc atgtcgctat gtgttctggg 481 aaatcaccat aaacgtgaaa tccctatcag tgatagagac ttataagttc cctatcagtg 541 atagagacgc tagcgtgttg taaatgagca cacaaaatac acatgctaaa atattatatt 601 ctatgacctt tataaaatca accaaaatct tctttttaat aactttagta tcaataatta 661 gaatttttat gttccttttt gcaaactttt aataaaaatg agcaaaataa aaaaacgcta 721 gttttagtaa ctcgcgttgt tttcttcacc tttaataata gctactccac cacttgaatt 781 ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg 841 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga 901 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga 961 tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc agccctgggt 1021 ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc gccgaccctg 1081 ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc gctacccttg 1141 tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg 1201 cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc gcagacggac 1261 agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat agcggctgct 1321 cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg gggtgtgggg 1381 cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag cctccggagc 1441 gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc cagggggatc 1501 tgtgagtttg gggacccttg attgttcttt ctttttcgct attgtaaaat tcatgttata 1561 tggagggggc aaagttttca gggtgttgtt tagaatggga agatgtccct tgtatcacca 1621 tggaccctca tgataatttt gtttctttca ctttctactc tgttgacaac cattgtctcc 1681 tcttattttc ttttcatttt ctgtaacttt ttcgttaaac tttagcttgc atttgtaacg 1741 aatttttaaa ttcacttttg tttatttgtc agattgtaag tactttctct aatcactttt 1801 ttttcaaggc aatcagggta tattatattg tacttcagca cagttttaga gaacaattgt 1861 tataattaaa tgataaggta gaatatttct gcatataaat tctggctggc gtggaaatat 1921 tcttattggt agaaacaact acatcctggt catcatcctg cctttctctt tatggttaca 1981 atgatataca ctgtttgaga tgaggataaa atactctgag tccaaaccgg gcccctctgc 2041 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt 2101 gctgtctcat cattttggca aagaattgta atacgactca ctatagggcg aattgatatg 2161 tctagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga 2221 atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg 2281 tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat 2341 aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat 2401 aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta 2461 ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc 2521 caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact 2581 ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct 2641 actactgata gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt 2701 gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt 2761 aaatgtgaaa gtgggtccgc gtacagcggc tcccgggagt tctagggatc tgcccctctc 2821 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 2881 tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 2941 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 3001 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 3061 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 3121 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 3181 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 3241 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 3301 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg 3361 ttttcctttg aaaaacacga tgataaggat ccaccggagc ttaccatgac cgagtacaag 3421 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 3481 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 3541 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 3601 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 3661 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 3721 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 3781 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 3841 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 3901 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 3961 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac 4021 ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg 4081 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg 4141 ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta 4201 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa 4261 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac 4321 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt 4381 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac 4441 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 4501 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 4561 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca 4621 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 4681 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga 4741 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca 4801 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 4861 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 4921 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 4981 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5041 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5101 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 5161 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5221 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5281 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 5341 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt 5401 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 5461 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 5521 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 5581 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 5641 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 5701 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 5761 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 5821 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 5881 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 5941 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6001 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6061 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6121 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6181 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6241 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6301 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 6361 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 6421 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 6481 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 6541 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 6601 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 6661 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 6721 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 6781 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 6841 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 6901 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 6961 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7021 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7081 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7141 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7201 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 7261 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 7321 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 7381 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 7441 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 7501 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc 7561 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt 7621 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac 7681 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct 7741 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt 7801 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc 7861 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 7921 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 7981 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga 8041 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc 8101 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg 8161 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc 8221 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat 8281 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc 8341 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga 8401 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat 8461 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac 8521 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga 8581 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca 8641 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc 8701 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac 8761 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg 8821 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa 8881 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg 8941 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttgga //