LOCUS Exported 7645 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Tet on shKras for conditional Kras knockdown. ACCESSION . VERSION . KEYWORDS TGMP.shKras.1422 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7645) AUTHORS Shao DD, Xue W, Krall EB, Bhutkar A, Piccioni F, Wang X, Schinzel AC, Sood S, Rosenbluh J, Kim JW, Zwang Y, Roberts TM, Root DE, Jacks T, Hahn WC TITLE KRAS and YAP1 Converge to Regulate EMT and Tumor Survival. JOURNAL Cell. 2014 Jul 3;158(1):171-84. doi: 10.1016/j.cell.2014.06.004. Epub 2014 Jun 19. PUBMED 24954536 REFERENCE 2 (bases 1 to 7645) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7645 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 579..920 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" CDS 987..1403 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 1333..1355 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 1371..1387 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" protein_bind 1451..1721 /label=tetracycline response element /bound_moiety="tetracycline repressor TetR" /note="contains seven copies of the tetracycline operator tetO" promoter 1754..1791 /label=minimal CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1788..1812 /label=LNCX /note="Human CMV promoter, forward primer" CDS 1885..2604 /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 1885..2601 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1930..1951) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2191..2210) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2538..2559 /label=EGFP-C /note="EGFP, forward primer" ncRNA 2648..2769 /label=5' miR-30a /note="sequence upstream of the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" ncRNA 2797..2811 /label=miR-30a loop /note="loop from the the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" ncRNA 2839..2965 /label=3' miR-30a /note="sequence downstream of the 71-nt precursor of the human miR-30a microRNA (Zeng et al., 2002)" /ncRNA_class="miRNA" promoter 2979..3478 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(3014..3037) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3411..3430 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3499..4098 /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(3499..3518) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3995..4015 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(4690..4706) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4690..4706) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4703..4725) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4714..4730 /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)." promoter complement(4738..4768) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4783..4804 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4921..4938) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5092..5680) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5172..5191) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5851..6711) /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 6474..6493 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6712..6816) /gene="bla" /label=AmpR promoter primer_bind 6884..6902 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6940..6962) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 7062..7081 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7275..7297 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7425..7444 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" LTR 7644..515 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" ORIGIN 1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc 301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc 361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata 421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca 481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga 541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg 601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt 661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa 721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga 781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag 841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa 901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg 961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct 1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga 1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag 1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc 1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc 1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg 1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag 1381 ccctcactcc ttctctaggc gccggaatta gatctggggg atcgatcctc tagagcatcg 1441 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc 1501 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga 1561 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc 1621 cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa 1681 aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagctcg 1741 gtacccgggt cgagtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 1801 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg 1861 gaccgatcca gcctctcgac caccatggtg agcaagggcg aggagctgtt caccggggtg 1921 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 1981 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 2041 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 2101 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 2161 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 2221 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 2281 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 2341 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 2401 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 2461 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 2521 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 2581 ggcatggacg agctgtacaa gtaatgacgt acggctaagc ctcgactagg gataacaggg 2641 taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca 2701 cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt atattgctgt 2761 tgacagtgag cgacagaccc agtatgaaat agtatagtga agccacagat gtatactatt 2821 tcatactggg tctgctgcct actgcctcgg aattcaaggg gctactttag gagcaattat 2881 cttgtttact aaaactgaat accttgctat ctctttgata catttttaca aagctgaatt 2941 aaaatggtat aaattaaatc acttttttca attctaccgg gtaggggagg cgcttttccc 3001 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 3061 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 3121 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 3181 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 3241 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 3301 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 3361 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 3421 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac 3481 ctgcagccca agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 3541 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 3601 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 3661 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 3721 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 3781 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg 3841 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 3901 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc 3961 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 4021 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 4081 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca 4141 cgaccccatg catcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg 4201 aaagacccca cctgtaggtt tggcaactag agaaccatca gatgtttcca gggtgcccca 4261 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct 4321 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg 4381 ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac cctcttgcag 4441 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac 4501 ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca acattctgag 4561 ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga atccacatac 4621 tccaatactc ctgaaatagt tcattatgga cagcgcagaa gagctgggga gaattaattc 4681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4741 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 4801 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4861 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 4921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 5101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 5161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 5461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5521 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 5761 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 5821 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 5881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5941 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 6001 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 6181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 6781 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 6841 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 6901 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 6961 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 7021 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 7081 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 7141 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 7201 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 7261 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc 7321 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa 7381 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata 7441 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag 7501 aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact 7561 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta 7621 tttagtctcc agaaaaaggg gggaa //