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
//
