LOCUS Exported 7770 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Cre-recombinase, a barcoded HDR template to introduce a G13D mutation into the endogenous Kras locus, and a Kras-targeting sgRNA to enhance HDR. . ACCESSION . VERSION . KEYWORDS AAV-KrasG13D/sgKras/Cre SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7770) AUTHORS Winters IP, Chiou SH, Paulk NK, McFarland CD, Lalgudi PV, Ma RK, TITLE Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity JOURNAL Nature Communications 8: 2053 (2017) REFERENCE 2 (bases 1 to 7770) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7770 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" regulatory 192..201 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter 2226..2466 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2226..2246 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2397..2416 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2495..2570 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(2658..2681) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3055..3074 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3126..3146 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3147..4175 /codon_start=1 /product="site-specific recombinase" /label=Cre /note="Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences." /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED GD" repeat_region 4476..4616 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4476..4605 /label=AAV2 ITR primer_bind complement(4655..4672) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4655..4671) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4664..4686) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4884..5339 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4971..4990) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5181..5202 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5356..5375) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5475..5497 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5535..5553) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5621..5725 /gene="bla" /label=AmpR promoter CDS 5726..6586 /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 LIKYW" primer_bind complement(5944..5963) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6757..7345 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7246..7265 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7499..7516 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7633..7654 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7669..7699 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7707..7723 /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 7712..7734 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7731..7747 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7731..7747 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct ggaggggtgg agtcgtgacg taaagatctg atatcatcga tcgcgatgca 181 ttaattaagc ggccgccatg gcagttcttt tgtatccatt tgtctcttta tctgcaataa 241 tatatgttct acaagggaaa ggagataaag tgtatatatt aattttattt caaaaatgtt 301 tggaaaagca gactaggaaa caacagtgtt gtattcaggg ctgttaactg tatacagtgc 361 cagcacttag tgcacctgtt gggagtgcag tgtattataa gcaaggcaga agtcacagag 421 gttactgaga aactctttta tgttctttac attttttttc ttgccaaaaa tagtctttat 481 ctctgaattt cattcaattt tatttccttg tacagaggta ctgtttttag tactcaaagg 541 attatatgtt aaaattgatc tgatcctata ccaaatattt aaacccattt cccacagata 601 gtttatagat taaaatgcca tcaaatatta tctttatttg cttcattgcc taaatatgtc 661 tttccccagc acagtgcagt tttgacacca gcttcggctt cctattttgt tgccctttat 721 tgcagaactg ctctgatggc tttaaaacag ttctctgcag ttgttggctc caacacagat 781 gttcttaggc tacctaactt ctaactttta atatccagtc aacaaagaat accgcaaggg 841 taggtgttgg gatagctgtc gacaagctca tgcgggtgtg tccacagggt atagcgtact 901 atgcagaata tttgtactga gtgaagtcat gatacattcc tttgagagcc attagctgct 961 acaaaacagt aatctggctg tttagatcaa caagctaaat gatagaagat gaaagtactg 1021 gtttccatgt atttttatta agtgttgatg agaaagttgt aagtgactta caggttactc 1081 tgtacatctg tagtcactga attcggaata tcttagagtt ttacacacct aggtgagtgt 1141 taaaatattg ataaagtttt tgataatctt gtgtgagaca tgttctaatt tagttgtatt 1201 ttattatttt tattgtaagg cctgctgaaa atgactgagt ataaactagt agtcgttgga 1261 gctggtgacg tgggtaagtc agcgctgaca atacagctaa ttcagaatca ctttgtggat 1321 gagtatgacc ctacgataga ggtaacgctg ctctacagtc tgcgtacgct tgtaaaggac 1381 ggcagccagc cgctttgaaa aagatatcat ttttatattt attagaaaat tatattgaaa 1441 gttatttcag ttatatgtga tgtcctttag ttccaaggct ttaaactggg tgttagggaa 1501 ccataggtgc aagaaagtcc acttctcatg agagctcacc acagagaaag aaagtccact 1561 tctcatgaga gcttaccaca gagagtaagg tccaagatgc catccgtgct ttgtatttgc 1621 attttgataa ataaatttca tgattttgtg agagttaaga atatagcttt gcctttgatc 1681 tattcctgaa aggtttgttg ttgttttatg ggtttgttgt tattgtgttg ttgtttggtg 1741 tcttttaggc agggtcttac tatgcagccc tgaccggcct agaactcaac tatgtagacc 1801 agactagctc tgaattctca gagatccatc tacctgcctc tgctgcccag atgctgcgat 1861 taaagatgta caccaccatg tacagtgaaa gttactcttt tgaaaacact ttattgttat 1921 agattacttg gaatgttgat gtgatgagaa aatttgatta tactaggaaa atagaatcac 1981 ttgatggttg agcttttttt ttaagactta tttattttat gtaagtgagt acactgcttc 2041 tctcttcaga cacaccagaa gagggcatca gatcccatta agatggctgt gagccaccat 2101 gtggttgctg ggaattgaac tcaggacctc tggaaaggca gtcagtgctc tttttttgtt 2161 taaagattta tttattttta tgaatacagt gtcactgtct tcatacacac aagagctcga 2221 gctgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta 2281 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac 2341 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact 2401 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga 2461 aaggaccaaa caccgactga gtataaactt gtgggtttta gagctagaaa tagcaagtta 2521 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt 2581 ggcgcgccta tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt 2641 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa 2701 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct 2761 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc 2821 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc 2881 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc 2941 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc 3001 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct 3061 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg 3121 ggatgcccaa gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg 3181 cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg 3241 atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg 3301 cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg 3361 attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg 3421 gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg 3481 tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac 3541 aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg 3601 atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac 3661 gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa 3721 tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac 3781 ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg 3841 atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca 3901 ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt 3961 acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg 4021 tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg 4081 gtggctggac caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag 4141 gggcaatggt gcgcctgctg gaagatggcg attagccatg tttaagggtt cctctagtgt 4201 acacgcgttg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 4261 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 4321 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 4381 gaggattggg aagacaatag caggcatgct ggggaacgcg tgatatcaga tctgttacgt 4441 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc 4501 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg 4561 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggtcga 4621 ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg 4681 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 4741 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 4801 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4861 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 4921 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc 4981 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 5041 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt 5101 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 5161 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat 5221 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa 5281 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt 5341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc 5401 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 5461 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 5521 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 5581 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 5641 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5701 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 5761 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5821 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 5881 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 5941 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 6001 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 6061 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 6121 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 6181 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 6241 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 6301 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 6361 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6421 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 6481 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 6541 tagacagatc gctgagatag gtgcctcact gattaagtat tggtaactgt cagaccaagt 6601 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 6661 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 6721 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 6781 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 6841 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 6901 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 6961 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 7021 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 7081 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 7141 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 7201 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 7261 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 7321 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 7381 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 7441 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 7501 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 7561 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 7621 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 7681 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 7741 ctatgaccat gattacgcca agcttgcatg //