LOCUS Exported 8323 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Constitutive expression of sgRNA targeting human TP53. ACCESSION . VERSION . KEYWORDS pXPR003-sgTP53-3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8323) AUTHORS Giacomelli AO, Yang X, Lintner RE, McFarland JM, Duby M, Kim J, Howard TP, Takeda DY, Ly SH, Kim E, Gannon HS, Hurhula B, Sharpe T, Goodale A, Fritchman B, Steelman S, Vazquez F, Tsherniak A, Aguirre AJ, Doench JG, Piccioni F, Roberts CWM, Meyerson M, Getz G, Johannessen CM, Root DE, Hahn WC TITLE Mutational processes shape the landscape of TP53 mutations in human cancer. JOURNAL Nat Genet. 2018 Oct;50(10):1381-1387. doi: 10.1038/s41588-018-0204-y. PUBMED 30224644 REFERENCE 2 (bases 1 to 8323) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8323 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..181 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 228..353 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 846..1079 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1264..1308 /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" promoter 1486..1726 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1486..1506 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1657..1676 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1756..1831 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 1887..2004 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2143..3321 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2374..3312 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3269..3289 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 3334..3933 /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(3334..3353) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3830..3850 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 3949..4537 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4002..4022) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4420..4431) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4609..4842 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4920..5041 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4957..4976) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5011..5030 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 5081..5216 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5143..5162 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5236..5255) /label=T7 /note="T7 promoter, forward primer" promoter complement(5237..5255) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5265..5282) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5265..5281) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5274..5296) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5423..5878 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5510..5529) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5720..5741 /label=F1ori-F /note="F1 origin, forward primer" promoter 5904..6008 /gene="bla" /label=AmpR promoter CDS 6009..6869 /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(6227..6246) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7040..7628 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7529..7548 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7782..7799 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7916..7937 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7952..7982 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7990..8006 /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 7995..8017 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8014..8030 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8014..8030 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 8049..8069 /label=T3 /note="T3 promoter, forward primer" promoter 8051..8069 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 8097..8323 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" ORIGIN 1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc 1501 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat 1561 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 1621 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 1681 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcccct 1741 tgccgtccca agcaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 1801 tcaacttgaa aaagtggcac cgagtcggtg cttttttaag cttggcgtaa ctagatcttg 1861 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 1921 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 1981 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg 2041 cgccggctcg agggggcccg ggtgcaaaga tggataaagt tttaaacaga gaggaatctt 2101 tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag 2161 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga 2221 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc 2281 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt 2341 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg 2401 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag 2461 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc 2521 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc 2581 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc 2641 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat 2701 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc 2761 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc 2821 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt 2881 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga 2941 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag 3001 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca 3061 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg 3121 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag 3181 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc 3241 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt 3301 cttccatttc aggtgtcgtg acgtacggcc accatgaccg agtacaagcc cacggtgcgc 3361 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 3421 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 3481 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 3541 ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 3601 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 3661 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga 3721 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 3781 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 3841 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 3901 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg 3961 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 4021 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 4081 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 4141 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 4201 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 4261 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 4321 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc 4381 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 4441 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 4501 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt 4561 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 4621 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc 4681 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 4741 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 4801 gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat 4861 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 4921 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 4981 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 5041 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc 5101 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 5161 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 5221 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 5281 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 5341 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 5401 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 5461 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 5521 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 5581 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 5641 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 5701 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 5761 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 5821 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 5881 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 5941 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 6001 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 6061 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 6121 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 6181 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 6241 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 6301 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 6361 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 6421 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 6481 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 6541 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 6601 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 6661 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 6721 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 6781 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 6841 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 6901 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 6961 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 7021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 7081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 7141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 7201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 7261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 7321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 7381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 7441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 7501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 7561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 7621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 7681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 7741 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 7801 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 7861 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 7921 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 7981 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 8041 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta 8101 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt 8161 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc 8221 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca 8281 ttgcagagat attgtattta agtgcctagc tcgatacata aac //