LOCUS Exported 9082 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral expression of NRAS. ACCESSION . VERSION . KEYWORDS pHAGE-NRAS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9082) AUTHORS Ng PK, Li J, Jeong KJ, Shao S, Chen H, Tsang YH, Sengupta S, Wang Z, Bhavana VH, Tran R, Soewito S, Minussi DC, Moreno D, Kong K, Dogruluk T, Lu H, Gao J, Tokheim C, Zhou DC, Johnson AM, Zeng J, Ip CKM, Ju Z, Wester M, Yu S, Li Y, Vellano CP, Schultz N, Karchin R, Ding L, Lu Y, Cheung LWT, Chen K, Shaw KR, Meric-Bernstam F, Scott KL, Yi S, Sahni N, Liang H, Mills GB TITLE Systematic Functional Annotation of Somatic Mutations in Cancer. JOURNAL Cancer Cell. 2018 Mar 12;33(3):450-462.e10. doi: 10.1016/j.ccell.2018.01.021. PUBMED 29533785 REFERENCE 2 (bases 1 to 9082) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9082 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..634 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 681..806 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1303..1536 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1721..1765 /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" misc_feature 2058..2175 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2223..3401 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2454..3392 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3349..3369 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 3421..3445 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(4019..4043) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 4096..4137 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" misc_feature 4158..4725 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" misc_feature 4175..4727 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4319..4336) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4546..4565 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4739..5458 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 4739..5455 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4784..4805) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5045..5064) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5392..5413 /label=EGFP-C /note="EGFP, forward primer" misc_feature 5472..6060 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5525..5545) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5943..5954) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6135..6368 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(6487..6505) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6573..6677 /gene="bla" /label=AmpR promoter CDS 6678..7538 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6896..6915) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7709..8297 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8198..8217 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8451..8468 /label=L4440 /note="L4440 vector, forward primer" primer_bind 8560..8579 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 8708..8729 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8744..8774 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8782..8798 /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 8787..8809 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8806..8822 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8806..8822 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 8872..8891 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg 2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt 2221 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 2281 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa 2341 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 2401 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 2461 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 2521 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt 2581 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct 2641 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg 2701 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct 2761 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg 2821 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc 2881 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc 2941 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac 3001 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga 3061 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc 3121 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg 3181 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga 3241 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt 3301 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga 3361 cagtggttca aagttttttt cttccatttc aggtgtcgtg aagcggccct gcagatatca 3421 acaagtttgt acaaaaaagc aggcaccatg actgagtaca aactggtggt ggttggagca 3481 ggtggtgttg ggaaaagcgc actgacaatc cagctaatcc agaaccactt tgtagatgaa 3541 tatgatccca ccatagagga ttcttacaga aaacaagtgg ttatagatgg tgaaacctgt 3601 ttgttggaca tactggatac agctggacaa gaagagtaca gtgccatgag agaccaatac 3661 atgaggacag gcgaaggctt cctctgtgta tttgccatca ataatagcaa gtcatttgcg 3721 gatattaacc tctacaggga gcagattaag cgagtaaaag actcggatga tgtacctatg 3781 gtgctagtgg gaaacaagtg tgatttgcca acaaggacag ttgatacaaa acaagcccac 3841 gaactggcca agagttacgg gattccattc attgaaacct cagccaagac cagacagggt 3901 gttgaagatg ctttttacac actggtaaga gaaatacgcc agtaccgaat gaaaaaactc 3961 aacagcagtg atgatgggac tcagggttgt atgggattgc catgtgtggt gatgtagaac 4021 ccagctttct tgtacaaagt ggttgatatc cagcacagtg gcggccgctc gagtctagag 4081 ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt 4141 accggttagt aatgagatcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa 4201 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat 4261 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct 4321 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct 4381 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc 4441 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa 4501 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc 4561 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg 4621 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc 4681 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg ccacacatat 4741 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 4801 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 4861 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 4921 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 4981 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 5041 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 5101 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 5161 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 5221 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 5281 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 5341 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 5401 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaat 5461 cgatagatcc taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta 5521 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta 5581 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt 5641 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg 5701 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt 5761 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag 5821 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc 5881 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc 5941 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc 6001 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc 6061 ctgagatcct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa 6121 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc 6181 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg 6241 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg 6301 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat 6361 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg 6421 aatatcagag agtgagaggc ccgggttaat taaggaaagg gctagatcat tcttgaagac 6481 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 6541 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 6601 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 6661 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 6721 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 6781 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 6841 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 6901 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact 6961 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 7021 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 7081 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 7141 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 7201 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 7261 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 7321 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 7381 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 7441 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 7501 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 7561 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 7621 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 7681 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 7741 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 7801 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 7861 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 7921 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 7981 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 8041 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 8101 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 8161 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 8221 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 8281 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 8341 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 8401 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 8461 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 8521 ttcattaatg cagcaagctc atggctgact aatttttttt atttatgcag aggccgaggc 8581 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 8641 ttgcaaaaag ctccccgtgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 8701 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 8761 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 8821 acatgattac gaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 8881 ccaaactcat caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa 8941 atcatcccaa acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt 9001 tactctaaac ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa 9061 tatgtactac aaacttagta gt //