LOCUS Exported 7942 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Flag pLJM1 Rap2A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7942) 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 7942) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7942 /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 1025..1142 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1191..1701 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1620..1638 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 1723..2322 /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(1723..1742) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2219..2239 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 2450..2683 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2755..2876 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2792..2811) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2846..2865 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 2916..3051 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2978..2997 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3071..3090) /label=T7 /note="T7 promoter, forward primer" promoter complement(3072..3090) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3100..3117) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3100..3116) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3109..3131) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3258..3713 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3345..3364) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3555..3576 /label=F1ori-F /note="F1 origin, forward primer" promoter 3739..3843 /gene="bla" /label=AmpR promoter CDS 3844..4704 /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(4062..4081) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4875..5463 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5364..5383 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5617..5634 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5751..5772 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5787..5817 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5825..5841 /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 5830..5852 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5849..5865 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5849..5865 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5884..5904 /label=T3 /note="T3 promoter, forward primer" promoter 5886..5904 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 5932..6158 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6159..6339 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6386..6511 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7004..7237 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7422..7466 /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 7764..7784 /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 cgatgcgcga gtacaaagtg gtggtgctgg gctcgggcgg ggtaggcaaa tccgccctga 481 ccgtgcagtt cgtgaccggc accttcatcg agaaatacga ccccaccatc gaggacttct 541 accgcaagga gatcgaggtg gattcgtcgc cgtcggtgct ggagatcctg gacacggcgg 601 gcaccgagca gttcgcgtcc atgcgggacc tgtacatcaa gaacggccag ggcttcatcc 661 tcgtctacag cctcgtcaac cagcagagct tccaggacat caagcccatg cgggaccaga 721 tcatccgcgt gaagcggtat gagaaagtgc cagtcatctt ggttgggaac aaagtggacc 781 tggaaagtga gagagaagta tcgtccagcg aaggcagagc ccttgctgaa gagtggggct 841 gcccctttat ggaaacttcc gctaagagta aaacaatggt ggacgaactc tttgcagaaa 901 ttgtgaggca gatgaactat gctgctcagc ctgacaaaga tgacccatgc tgttctgcat 961 gtaacataca ataggatatc ttcgaattct cgacctcgag acaaatggca gtattcatcc 1021 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca 1081 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt 1141 ttcgggttta ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg 1201 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt 1261 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc 1321 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc 1381 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca 1441 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg 1501 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa 1561 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg 1621 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga 1681 atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc 1741 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg 1801 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc 1861 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 1921 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 1981 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 2041 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 2101 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 2161 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 2221 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 2281 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg 2341 cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact 2401 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 2461 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac 2521 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa 2581 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag 2641 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat 2701 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg 2761 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 2821 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 2881 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 2941 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 3001 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt 3061 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg 3121 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 3181 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 3241 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3301 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3361 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 3421 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3481 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3541 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3601 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 3661 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc 3721 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 3781 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 3841 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 3901 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 3961 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4021 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4081 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4141 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4201 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 4261 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 4321 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 4381 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 4441 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 4501 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 4561 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 4621 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 4681 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 4741 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 4801 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 4861 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 4921 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 4981 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 5041 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5101 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5161 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5221 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5281 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5341 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 5401 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 5461 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 5521 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 5581 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 5641 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 5701 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 5761 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 5821 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 5881 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt 5941 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag 6001 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat 6061 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca 6121 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga 6181 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 6241 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 6301 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt 6361 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg 6421 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg 6481 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg 6541 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt 6601 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga 6661 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact 6721 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa 6781 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc 6841 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa 6901 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg 6961 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg 7021 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg 7081 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg 7141 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc 7201 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa 7261 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac 7321 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct 7381 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat 7441 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt 7501 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg 7561 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc 7621 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag 7681 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc 7741 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata 7801 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 7861 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 7921 tatgttttaa aatggactat ca //