LOCUS Exported 8431 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Flag pLJM1 RagB 99L SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8431) AUTHORS Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM TITLE The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. JOURNAL Science. 2008 Jun 13. 320(5882):1496-501. PUBMED 18497260 REFERENCE 2 (bases 1 to 8431) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8431 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 131..334 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 285..305 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 375..398 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 1514..1631 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1680..2190 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2109..2127 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2212..2811 /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(2212..2231) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2708..2728 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 2939..3172 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3244..3365 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3281..3300) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3335..3354 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3405..3540 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3467..3486 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3560..3579) /label=T7 /note="T7 promoter, forward primer" promoter complement(3561..3579) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3589..3606) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3589..3605) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3598..3620) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3747..4202 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3834..3853) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4044..4065 /label=F1ori-F /note="F1 origin, forward primer" promoter 4228..4332 /gene="bla" /label=AmpR promoter CDS 4333..5193 /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(4551..4570) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5364..5952 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5853..5872 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6106..6123 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6240..6261 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6276..6306 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6314..6330 /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 6319..6341 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6338..6354 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6338..6354 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6373..6393 /label=T3 /note="T3 promoter, forward primer" promoter 6375..6393 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6421..6647 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6648..6828 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6875..7000 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7493..7726 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7911..7955 /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" primer_bind 8253..8273 /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta 361 gcgctaccgg tatggactac aaggatgacg atgacaaggg tggtggtggt ggtgcgtcga 421 cgatggaaga atctgactct gagaaaacga cggagaaaga aaatctgggg ccgagaatgg 481 atccaccact aggggaaccg gaaggatcgc ttgggtgggt gctaccaaat acagccatga 541 agaaaaaggt gctgttgatg ggtaaaagtg ggtctggtaa gaccagcatg aggtctatta 601 tctttgcaaa ttatattgcc agagacacac gtcgccttgg cgcaacaatt gatgtagaac 661 attctcatgt tcgatttctg ggaaacctgg tattgaacct gtgggattgt ggtgggctag 721 acaccttcat ggaaaattat ttcactagcc aacgggacaa catcttccga aatgtggagg 781 ttctgattta tgtctttgat gtggagagcc gcgaactgga aaaggacatg cactattacc 841 aatcatgcct ggaggccatt ctgcagaatt ctccagatgc caaaatattt tgcttggtac 901 acaaaatgga tctggtacag gaggatcaac gggacctgat ttttaaagag cgagaagaag 961 atttgaggcg tttgtctcgc ccattggaat gttcttgttt ccgaacatct atctgggatg 1021 aaaccctcta taaggcttgg tccagcatag tttatcagct gattcccaat gttcagcagc 1081 tggaaatgaa cctaaggaat tttgctgaaa ttatcgaagc tgatgaagta cttctttttg 1141 agagagctac ttttctggta atttctcact atcagtgtaa agagcagcgt gatgcccata 1201 gatttgagaa aataagcaac attattaagc agttcaagct gagctgcagc aagctggctg 1261 cctctttcca gagtatggaa gtcaggaact ctaacttcgc tgctttcatt gacatcttta 1321 catccaacac ttatgtgatg gttgtgatgt ctgatccgtc cattccttct gcagctactc 1381 tgatcaacat ccgcaatgcc aggaaacact ttgaaaagct ggaaagagtg gatggaccaa 1441 agcagtgtct tctcatgcgc taagatatct tcgaattctc gacctcgaga caaatggcag 1501 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa 1561 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa 1621 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg 1681 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct 1741 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc 1801 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct 1861 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg 1921 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg 1981 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc 2041 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg 2101 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc 2161 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag 2221 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc 2281 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc 2341 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag 2401 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa 2461 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg 2521 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg 2581 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc 2641 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc 2701 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag 2761 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc 2821 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga 2881 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 2941 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc 3001 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 3061 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 3121 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt 3181 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag 3241 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 3301 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 3361 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc 3421 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 3481 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 3541 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 3601 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 3661 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 3721 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg 3781 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 3841 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 3901 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 3961 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 4021 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 4081 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 4141 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat 4201 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 4261 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4321 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 4381 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 4441 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 4501 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 4561 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 4621 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 4681 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 4741 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 4801 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 4861 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 4921 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 4981 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5041 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5101 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5161 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5221 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 5281 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 5341 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 5401 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 5461 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 5521 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 5581 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 5641 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 5701 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 5761 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 5821 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 5881 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 5941 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 6001 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 6061 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 6121 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 6181 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 6241 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 6301 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 6361 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa 6421 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 6481 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 6541 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc 6601 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt 6661 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 6721 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 6781 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 6841 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc 6901 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 6961 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 7021 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 7081 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 7141 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 7201 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 7261 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 7321 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 7381 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 7441 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 7501 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga 7561 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 7621 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 7681 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 7741 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 7801 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt 7861 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac 7921 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt 7981 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag 8041 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc 8101 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg 8161 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac 8221 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat 8281 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca 8341 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag 8401 ttttaaaatt atgttttaaa atggactatc a //