LOCUS Exported 8431 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Flag pLJM1 RagB 54L 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 1..204 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 155..175 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 245..268 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 1384..1501 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1550..2060 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1979..1997 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2082..2681 /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(2082..2101) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2578..2598 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 2809..3042 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3114..3235 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3151..3170) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3205..3224 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3275..3410 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3337..3356 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3430..3449) /label=T7 /note="T7 promoter, forward primer" promoter complement(3431..3449) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3459..3476) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3459..3475) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3468..3490) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3617..4072 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3704..3723) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3914..3935 /label=F1ori-F /note="F1 origin, forward primer" promoter 4098..4202 /gene="bla" /label=AmpR promoter CDS 4203..5063 /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(4421..4440) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5234..5822 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5723..5742 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5976..5993 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6110..6131 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6146..6176 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6184..6200 /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 6189..6211 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6208..6224 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6208..6224 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6243..6263 /label=T3 /note="T3 promoter, forward primer" promoter 6245..6263 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6291..6517 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6518..6698 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6745..6870 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7363..7596 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7781..7825 /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 8123..8143 /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg 241 tatggactac aaggatgacg atgacaaggg tggtggtggt ggtgcgtcga cgatggaaga 301 atctgactct gagaaaacga cggagaaaga aaatctgggg ccgagaatgg atccaccact 361 aggggaaccg gaaggatcgc ttgggtgggt gctaccaaat acagccatga agaaaaaggt 421 gctgttgatg ggtaaaagtg ggtctggtaa gctcagcatg aggtctatta tctttgcaaa 481 ttatattgcc agagacacac gtcgccttgg cgcaacaatt gatgtagaac attctcatgt 541 tcgatttctg ggaaacctgg tattgaacct gtgggattgt ggtgggcaag acaccttcat 601 ggaaaattat ttcactagcc aacgggacaa catcttccga aatgtggagg ttctgattta 661 tgtctttgat gtggagagcc gcgaactgga aaaggacatg cactattacc aatcatgcct 721 ggaggccatt ctgcagaatt ctccagatgc caaaatattt tgcttggtac acaaaatgga 781 tctggtacag gaggatcaac gggacctgat ttttaaagag cgagaagaag atttgaggcg 841 tttgtctcgc ccattggaat gttcttgttt ccgaacatct atctgggatg aaaccctcta 901 taaggcttgg tccagcatag tttatcagct gattcccaat gttcagcagc tggaaatgaa 961 cctaaggaat tttgctgaaa ttatcgaagc tgatgaagta cttctttttg agagagctac 1021 ttttctggta atttctcact atcagtgtaa agagcagcgt gatgcccata gatttgagaa 1081 aataagcaac attattaagc agttcaagct gagctgcagc aagctggctg cctctttcca 1141 gagtatggaa gtcaggaact ctaacttcgc tgctttcatt gacatcttta catccaacac 1201 ttatgtgatg gttgtgatgt ctgatccgtc cattccttct gcagctactc tgatcaacat 1261 ccgcaatgcc aggaaacact ttgaaaagct ggaaagagtg gatggaccaa agcagtgtct 1321 tctcatgcgc taagatatct tcgaattctc gacctcgaga caaatggcag tattcatcca 1381 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1441 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt 1501 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt 1561 tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt 1621 ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg 1681 tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc 1741 taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac 1801 gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc 1861 gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag 1921 gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt 1981 gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa 2041 tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag tacaagccca 2101 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 2161 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 2221 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 2281 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 2341 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 2401 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 2461 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 2521 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 2581 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 2641 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc 2701 agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt 2761 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 2821 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc 2881 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 2941 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 3001 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc 3061 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt 3121 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3181 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 3241 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3301 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3361 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta 3421 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt 3481 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 3541 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 3601 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 3661 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 3721 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 3781 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 3841 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 3901 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 3961 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 4021 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca 4081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 5041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 5101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 5161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 5221 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 5281 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 5341 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 5401 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 5461 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 5521 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 5581 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 5641 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 5701 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 5761 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 5821 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5881 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5941 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 6001 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 6061 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 6121 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 6181 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 6241 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta 6301 tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg 6361 agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt 6421 aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag 6481 agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat 6541 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 6601 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 6661 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg 6721 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc 6781 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc 6841 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga 6901 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta 6961 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa 7021 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt 7081 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa 7141 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca 7201 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 7261 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 7321 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 7381 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 7441 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 7501 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 7561 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 7621 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 7681 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 7741 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 7801 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 7861 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 7921 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 7981 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 8041 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc 8101 tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat 8161 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt 8221 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt 8281 atgttttaaa atggactatc atatgccaag tacgccccct attgacgtca atgacggtaa 8341 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 8401 catctacgta ttagtcatcg ctattaccat g //