LOCUS Exported 8083 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION 3rd gen lentiviral vector for EGFP fusion; PGK driven puromycin. ACCESSION . VERSION . KEYWORDS pLJM1-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8083) 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 8083) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8083 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 290..493 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 444..464 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 532..541 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 538..1254 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 538..1254 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(583..604) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(844..863) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1191..1212 /label=EGFP-C /note="EGFP, forward primer" misc_feature 1325..1442 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1491..2001 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 1920..1938 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2023..2622 /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(2023..2042) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2519..2539 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 2750..2983 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3055..3176 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3092..3111) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3146..3165 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3216..3351 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3278..3297 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3371..3390) /label=T7 /note="T7 promoter, forward primer" promoter complement(3372..3390) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3400..3417) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3400..3416) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3409..3431) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3558..4013 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3645..3664) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3855..3876 /label=F1ori-F /note="F1 origin, forward primer" promoter 4039..4143 /gene="bla" /label=AmpR promoter CDS 4144..5004 /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(4362..4381) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5175..5763 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5664..5683 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5917..5934 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6051..6072 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6087..6117 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6125..6141 /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 6130..6152 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6149..6165 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6149..6165 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6184..6204 /label=T3 /note="T3 promoter, forward primer" promoter 6186..6204 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6232..6458 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6459..6639 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6686..6811 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7304..7537 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7722..7766 /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 8064..1 /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 61 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 121 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgccaagta cgccccctat 181 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 241 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 301 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 361 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 421 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 481 tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg 541 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 601 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 661 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 721 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 781 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 841 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 901 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 961 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 1021 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 1081 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 1141 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 1201 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga 1261 ctcagatctc gagctcaagc ttcgaattct cgacctcgag acaaatggca gtattcatcc 1321 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca 1381 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt 1441 ttcgggttta ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg 1501 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt 1561 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc 1621 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc 1681 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca 1741 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg 1801 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa 1861 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg 1921 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga 1981 atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc 2041 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg 2101 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc 2161 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 2221 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 2281 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 2341 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 2401 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 2461 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 2521 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 2581 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg 2641 cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact 2701 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 2761 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac 2821 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa 2881 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag 2941 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat 3001 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg 3061 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 3121 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 3181 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 3241 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 3301 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt 3361 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg 3421 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 3481 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 3541 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3601 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3661 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 3721 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3781 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3841 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 3901 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 3961 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc 4021 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 4081 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 4141 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 4201 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4261 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4321 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4381 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4441 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4501 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 4561 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 4621 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 4681 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 4741 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 4801 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 4861 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 4921 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 4981 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 5041 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 5101 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5161 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5221 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5281 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 5341 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5401 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5461 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5521 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5581 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5641 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 5701 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 5761 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 5821 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 5881 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 5941 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 6001 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 6061 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 6121 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 6181 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt 6241 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag 6301 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat 6361 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca 6421 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga 6481 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 6541 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 6601 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt 6661 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg 6721 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg 6781 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg 6841 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt 6901 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga 6961 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact 7021 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa 7081 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc 7141 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa 7201 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg 7261 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg 7321 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg 7381 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg 7441 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc 7501 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa 7561 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac 7621 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct 7681 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat 7741 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt 7801 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg 7861 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc 7921 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag 7981 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc 8041 ctcgagcggc cgcccccttc accgagggcc tatttcccat gat //