LOCUS Exported 7085 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pLKO.1-TSC2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7085) AUTHORS Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH TITLE Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. JOURNAL Nat Cell Biol. 2007 Mar . 9(3):316-23. PUBMED 17277771 REFERENCE 2 (bases 1 to 7085) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7085 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..227 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 228..408 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 455..580 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1073..1306 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1491..1535 /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 1833..2073 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1833..1853 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2004..2023 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2179..2296 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2345..2855 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2774..2792 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2877..3476 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(2877..2896) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3373..3393 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 3604..3837 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3909..4030 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3946..3965) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4000..4019 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4070..4205 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4132..4151 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4225..4244) /label=T7 /note="T7 promoter, forward primer" promoter complement(4226..4244) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4254..4271) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4254..4270) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4263..4285) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4412..4867 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4499..4518) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4709..4730 /label=F1ori-F /note="F1 origin, forward primer" promoter 4893..4997 /gene="bla" /label=AmpR promoter CDS 4998..5858 /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(5216..5235) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6029..6617 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6518..6537 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6771..6788 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6905..6926 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6941..6971 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6979..6995 /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 6984..7006 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7003..7019 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7003..7019 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7038..7058 /label=T3 /note="T3 promoter, forward primer" promoter 7040..7058 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc 181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt 241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc 481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga 1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt 1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac 1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt 1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag 1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc 1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg 1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac 1801 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat 1861 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca 1921 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag 1981 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg 2041 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggtcactgg ccttggacgg 2101 tattgctcga gcaataccgt ccaaggccag tgtttttgaa ttctcgacct cgagacaaat 2161 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga 2221 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac 2281 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggct 2341 cgagggggtt ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct 2401 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca 2461 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc 2521 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac 2581 aaacggaagc cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg 2641 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga 2701 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt 2761 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct 2821 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccaccggag cttaccatga 2881 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca 2941 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc 3001 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg 3061 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg 3121 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc 3181 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg 3241 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg 3301 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga 3361 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg 3421 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc 3481 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcggtacctt 3541 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 3601 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 3661 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 3721 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 3781 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 3841 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga 3901 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 3961 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 4021 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct 4081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 4141 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 4201 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc 4261 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 4321 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 4381 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg 4441 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 4501 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 4561 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 4621 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 4681 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 4741 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 4801 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt 4861 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 4921 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4981 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 5041 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5101 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5161 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 5221 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 5281 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 5341 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 5401 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 5461 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5521 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 5581 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5641 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5701 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5761 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 5821 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 5881 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 5941 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 6001 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6061 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 6121 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 6181 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 6241 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 6301 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 6361 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 6421 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 6481 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 6541 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6601 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 6661 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 6721 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 6781 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 6841 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 6901 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 6961 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 7021 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag 7081 cttaa //